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Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism
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  • Manuel José Ruiz Munevar
    Manuel José Ruiz Munevar
    Laboratory of Molecular Modelling & Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
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  • Valerio Rizzi
    Valerio Rizzi
    Biomolecular & Pharmaceutical Modelling Group, Université de Genève, Rue Michel-Servet 1, Geneva CH-1211 4, Switzerland
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    Orcidhttps://orcid.org/0000-0001-5126-8996
  • Corinne Portioli
    Corinne Portioli
    Laboratory of Nanotechnology for Precision Medicine, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    Laboratory of Brain Development and Disease, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
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  • Pietro Vidossich
    Pietro Vidossich
    Laboratory of Molecular Modelling & Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
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  • Erhu Cao
    Erhu Cao
    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, United States
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  • Michele Parrinello*
    Michele Parrinello
    Laboratory of Atomistic Simulations, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    *Email: [email protected]
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  • Laura Cancedda*
    Laura Cancedda
    Laboratory of Brain Development and Disease, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    *Email: [email protected]
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  • Marco De Vivo*
    Marco De Vivo
    Laboratory of Molecular Modelling & Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-4022-5661
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2024, 146, 1, 552–566
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https://doi.org/10.1021/jacs.3c10258
Published December 25, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1’s water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1’s function modulation and thus to potential drug action on selected CCCs.

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Introduction

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The cation chloride cotransporter (CCC) NKCC1 is a modulator of intracellular Cl– concentration in diverse cell types in numerous body organs. Particularly, NKCC1 is expressed in parenchymal brain cells, where it regulates intraneuronal Cl– concentration which in turn is crucial for the modulation of the function of the neurotransmitter GABA, Gamma-aminobutyric acid. (1) In the majority of cell types, NKCC1 uses an inward-directed Na+ gradient to import Na+, K+, and Cl–, in a 1:1:2 stoichiometry. (2) NKCC1 is also highly expressed on the apical membrane of the choroid plexus, where it plays a major role in producing and regulating cerebrospinal fluid (CSF). Importantly, in recent years, extensive research has shown that an increased intracellular Cl– concentration in neurons is symptomatically related to multiple neuropathologies and also glioblastoma, along with the growing body of literature, showing NKCC1’s important pathological role in increased CSF production. (3−7) Accordingly, normalization of intracellular Cl– concentration and of CSF hypersecretion in the brain by modulation of CCC (including NKCC1) functions is considered a very promising strategy for neuroscience drug discovery. (3,7−10) A fundamental understanding of NKCC1’s structure, dynamics, and overall conformational motions for ion passage may therefore open new avenues for therapeutic interventions on many human disorders ranging from brain and hearing to kidney and cancer diseases.
Interestingly, 15 NKCC1 structures have recently been resolved alone or together with unselective inhibitors (e.g., the FDA-approved diuretic bumetanide). All these recent cryo-EM structures of NKCC1 have clarified several functional features of its multidomain system, (11−15) revealing a structural similarity to the LeuT-fold transporters. (2,16) In particular, NKCC1 is a homodimer, where each monomer is constructed by three main components: a conserved transmembrane (TM) domain composed of 12 helices organized in two inverted repeats of five-helix bundles, which contain all four ion binding sites (Figure 1A). Two additional intracellular domains are the disordered amino-terminal and the large carboxy-terminal domains, which are related to NKCC1 activation (Figure 1B). (13)

Figure 1

Figure 1. NKCC1’s structure embedded in the membrane. (A) 3D representation of full-length human NKCC1 (PDB 7MXO), with each monomer represented in dark and light gray. Each monomer is constructed by three main components: (i) conserved TM domain, composed of 12 helices, which contains all four ion binding sites, shown in green, blue and orange for Cl–, Na+, and K+ ions, respectively; (ii) disordered amino-terminal; and (iii) large carboxy-terminal domains. (13) All structures of NKCC1, and related CCC’s of the same family, (40) revealed that the TM helices are organized in two inverted repeats of five-helix bundles─also known as LeuT-fold. (2,16,17,21) (B) Schematic representation of NKCC1, showing its homodimeric structure with one monomer represented to highlight the transmembrane domains (TMs, dark gray); the other monomer represented to highlight the channel arrangement across the cell membrane (light brown); the two five-helix bundle inverted repeats (TM 1 to TM 5 and TM 6 to TM 10) and the dimeric interface (TM 11 and TM 12). The amino and carboxy-terminal domains are also highlighted.

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The NKCC1 structures recently resolved have also confirmed that NKCC1 operates transiting from conformations where the ion binding sites are exposed either to the extracellular or to the intracellular side of the membrane. (17) That is, NKCC1 must transit dynamically through two distinct conformational states: an outward open (OO) state, in which the transporter binds to ions from outside of the cell, and an inward open (IO) state, in which the protein releases the ions to the inside of the cell (Figure 2). However, the mechanism for the IO ↔ OO conformational transitions in NKCC1 is unclear.

Figure 2

Figure 2. Essential conformational transition for alternating accessibility of ion binding sites allows NKCC1 ion transport. (A) Schematic representation of outward open (left) and inward open (right) NKCC1 conformation. Ionic binding sites are exposed to the extra and intracellular side of the membrane, respectively. (B) In gray, atomic surface representation of outward open (left) and inward open (right) NKCC1 conformation. Ionic binding sites for Na+, K+, and Cl– are shown as colored surfaces (blue, orange, and green, respectively), and ions are represented as colored spheres. Outer (left) and inner (right) vestibules, open to the extra and intracellular sides of the membrane, are highlighted with discontinuous black lines.

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In this context, we have explored here different molecular mechanisms for IO ↔ OO conformational transitions in NKCC1 and ion transport in NKCC1 embedded in the membrane. To do so, we have used extensive classical equilibrium molecular dynamics (MD) simulations and deep-learning-guided enhanced sampling free energy calculations. (18,19) We also validated our in silico evidence by biological functional studies in cell cultures with targeted NKCC1 mutagenesis. Combining our molecular modeling and molecular biology experiments, we clarified the complex IO ↔ OO conformational transition in NKCC1 and revealed that it operates through a rocking-bundle mechanism. In addition, we have identified specific interhelical dynamical contacts that we have found to be fundamentally involved in the NKCC1’s transport cycle, favoring water diffusion through the transporter. Based on structural similarities and biochemical data analyses, we propose that our findings could be extended to all Na+-dependent CCCs.

Results

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NKCC1 Operates via a Rocking-Bundle Mechanism for Conformational Transitions

To investigate the mechanism of the NKCC1 ion transporter, we ran extensive classical MD simulations coupled to the on-the-fly probability enhanced sampling (OPES) method to handle elaborated deep-learning-derived collective variables (CVs) needed to capture complex dynamical phenomena (19,20) and accelerate a meaningful sampling of the underlying conformational space. Notably, these simulations of atomistic models embedded into the membrane were grounded on recently resolved human NKCC1 structures. Specifically, our realistic models were based on the structure of the IO state (13) and a partially loaded OO state of NKCC1 (Figure 2B). (15) We initially focused on NKCC1 model systems in which ions bound at the vestibules are absent (i.e., the ions’ external gates at the transporter, see Figure 2). This setup allowed us to focus our exploration on the mechanistic transitions between the IO ↔ OO NKCC1 conformations. As a result, we could sample 16 IO ↔ OO conformational transitions (Figure S4) and collected over ∼2 μs of trajectories from a total of 3 independent enhanced sampling simulations, in addition to ∼2.5 μs of equilibrium MD runs.
There are diverse alternating access mechanisms that could allow NKCC1 to shuttle ions inside the cell, passing across the cell membrane. (17,21) For example, the “rocking-bundle” or the “elevator” mechanism are equally plausible alternating mechanisms for ions transport across the membrane. Significantly, these distinctive alternating mechanisms differ in the active motions of specific TM helices, which must assist the dynamic passage of ions. The IO ↔ OO conformational transitions observed in our simulations revealed that NKCC1 operates through the “rocking-bundle mechanism”. (22) In particular, our computational evidence shows the exact protein motions for NKCC1’s function, clarifying which specific dynamics operates within the general ‘alternating access mechanism’. We found indeed that the inner and outer vestibules of NKCC1 alternate their accessibility using the rocking-bundle mechanism for NKCC1 characterized by the motion of TM helices TM 4 and TM 9. In fact, both TM 4 and TM 9 stably maintained their initial structure for over ∼1 μs in both IO and OO states during our equilibrium MD, with an average root-mean-square deviation (RMSD) of 0.68 ± 0.14 and 0.72 ± 0.11 Å, respectively. However, in all the 16 IO ↔ OO transitions (Figure 3B), these TM helices showed a concerted angular motion, with a rotation of at most 14° with respect to the intracellular extreme of TM 2 and TM 7 - both located within the static domain formed by TM 1, TM 2, TM 6 and TM 7 of NKCC1 (Figure 3A), which is used for structure alignment. The RMSD of TM 4 and TM 9, in the IO and OO states of the cryo-EM structures, was 4.21 Å (vs = 1.75 Å for the whole remaining TMs). This confirmed the concerted movement of these two TM helices to be in line with an angular motion typical of the rocking-bundle mechanism (Figure S1; (23)).

Figure 3

Figure 3. Rocking-bundle angular motion of specific NKCC1 TMs facilitates alternate accessibility of ion binding sites. (A) Schematic representation of NKCC1’s angular motion, defined as the change of the angle (α) between TM 4 and TM 9 (in green) and TM 2 and TM 7 (in red), during the conformational transition between the outward open state (bright green) and inward open state (dim green), calculated from the centers of mass of the backbone atoms from the extracellular and intracellular tip of TM 4 and TM 9 and the intracellular tip of TM 2 and TM 7 (in red). (B) Quantification of TM 4 and TM 9 angular motion represented by the angle α through 16 conformational transitions from OPES Explore simulations. The light brown horizontal bars represent the outward open and inward open average angle α ± 1SD calculated from 1 μs of equilibrium molecular dynamics (MD). Circles represent the starting point of each transition, whereas the triangles represent the end point of the same transition and its direction. Circles and triangles are colored depending on the NKCC1 conformation they represent (bright green for outward open and dim green for inward open).

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Interestingly, we found that TM 4 and TM 9 operate as a joint structural motif due to several hydrophobic interactions at their interface, in agreement with previous structural observations. (15) Here, we found that these hydrophobic interactions involved Ala 414, Val 417, Val 418 and Leu 421 from TM 4, while Phe 659, Leu 663 and Ile 666 from TM 9 (Figure 4). These interactions, statically present also in the cryo-EM structures, (11−15) were stably maintained during our equilibrium MD simulations, in both the IO and OO states. In addition, we observed the crucial involvement of TM 10, which was key to the NKCC1’s rocking-bundle mechanism in our simulations. TM 10 is connected by a short loop on the extracellular side to TM 9. This connection made TM 10 susceptible to conformational changes due to TM 4 and TM 9’s angular motion, when transiting from the IO to the OO state (and vice versa). In our simulations, this motion modulated the solvent accessibility to the outer vestibule of NKCC1. In detail, TM 10 rested at an angle of 151.2 ± 2.9° during our equilibrium MD of the IO state, while it conserved an angle of 169.2 ± 4.0° in the OO state throughout the equilibrium MD. However, during IO → OO transitions, TM 4 and TM 9’s angular motion was critical to drag the TM 10's extracellular tip outward, allowing the solvent to access the outer vestibule (Figure 5A). In the IO → OO transitions, TM 10 was dynamically straightened (again, from 151.2 ± 2.9 to 169.2 ± 4.0°). This broke the interatomic contacts at the TM 10-TM 6 interface (i.e., Asn 672 with Ile 493, Pro 676 with Ala 492 and Ser 679 with Pro 496, at the TM 10 and TM 6, respectively; Figure 6, inset on the top right). Notably, Pro 496 and Ser 679 interacted with water molecules, thus potentially assisting the flooding of water into the NKCC1’s outer vestibule. This mechanism is again in line with a rocking-bundle mechanism. In addition, in agreement with this evidence, during OO → IO transitions TM 4 and TM 9’s angular motion pushed TM 10's extracellular tip inward, therefore disabling solvent accessibility to the outer vestibule of NKCC1. Taken together, these results show that the inward ↔ outward motion of TM 10 modulates NKCC1 solvent accessibility, as observed in all 16 conformational transitions (Figure 5B).

Figure 4

Figure 4. Stabilization of the hydrophobic interface between TM 4 and TM 9 allows for their cooperative action. Representation of human NKCC1 embedded in the cell membrane, with TM 4 and TM 9 highlighted in bright yellow. Inset on the right: Higher magnification of the hydrophobic interface between TM 4 and TM 9 (highlighted by the oval), which allows for their cooperative angular motion. Relevant residues are shown as sticks with their atomic surfaces pictured in red (oxygen), blue (nitrogen), gray (carbon), and white (hydrogen).

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Figure 5

Figure 5. NKCC1 TM 10's corking motion modulates ion/water access to the outer vestibule. (A) Schematic representation of NKCC1 TM 10's corking motion, defined as the change of TM 10's (in green) intrahelical angle (θ), during the conformational transition between the outward open state (bright green) and inward open state (dim green), calculated from the centers of mass of the backbone atoms from TM 10’s intra and extracellular tips, and the backbone atoms where TM 10 bends. (B) Quantification of TM 10's corking motion represented by the angle θ through 16 conformational transitions from OPES Explore simulations. The light brown horizontal bars represent the outward open and inward open average angle θ ± 1SD calculated from 1 μs of equilibrium molecular dynamics (MD). Circles represent the starting point of each transition, whereas the triangles represent the end point of the same transition and its direction. Circles and triangles are colored depending on the NKCC1 conformation they represent (bright green for outward open and dim green for inward open).

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Figure 6

Figure 6. Interface between TM 10 and TM 6 highlights crucial interactions that determine accessibility of extracellular binding sites. Representation of human NKCC1 embedded in the cell membrane, with TM 10 and TM 6 highlighted in bright yellow. Inset on the top right: In the IO state, as shown by the light green schematic representation, interacting residues at the TM 10 and TM 6 interface are shown as sticks with their atomic surfaces (pictured in red─oxygen, blue─nitrogen, gray─carbon, and white─hydrogen), blocking solvent access to the outer vestibule. Inset on the bottom right: In the OO state, as shown by the light orange schematic representation, previously interacting residues at the TM 10 and TM 6 interface are now shown to be too far apart to form bonds.

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Next, we also investigated the possibility for NKCC1 to operate by the so-called “elevator mechanism”, which would be an alternative to the rocking-bundle one. The possibility of identifying an elevator mechanism was evaluated by measuring the vertical translation of TM 4 and TM 9 vs TM 2 and TM 7, which are part of the static domain. We found that TM 4 and TM 9 did not show any vertical translation during any of the conformational transitions, as their relative positions in the IO and OO state cryo-EM structures were not vertically translated. This is not what would be expected for an elevator mechanism (Figure S5). Altogether, our results indicate a rocking-bundle mechanism for NKCC1 function while excluding an elevator mechanism.
To test the functional importance of TM 10's motion and association with TM 6 as evidenced by our simulation data, we performed cell and molecular biology experiments in standard stable cell lines (HEK293 kidney cells) transfected with 4 diverse mutants (Ala490Trp, Leu664Ala, Asn665Ala and Ala668Trp) of mouse NKCC1. Specifically, the NKCC1 constructs were generated with 4 mutations on residues located at the interface between TM 10 and TM 6. These TM residues, all located at the outer vestibule (Figure 7A), were selected because of their involvement in the dynamic interaction network highlighted by our MD simulations for NKCC1 conformational transitions. Next, we performed in vitro Cl– influx assay (24−26) measurements on HEK293 cells transfected with one of the four mouse NKCC1 mutants, at the time.

Figure 7

Figure 7. Mutagenesis targeting residues from TM 10 and TM 6 highlight their functional relevance. (A) Representation of human NKCC1 embedded in the cell membrane, with TM 10 and TM 6 highlighted in bright yellow. Inset on the right: The interface between TM 10 and TM 6 where homologous mutated residues are shown as sticks and are highlighted in green surface. Namely, these residues are mouse A490W (human Ala 497), mouse L664A (human Leu 671), mouse N665A (human Asn 672), and mouse A668W (human Ala 675). Gray surface represents the position of residues that mainly form/break interactions throughout TM 10's corking motion. (B) Example traces obtained in the Cl– influx assay on HEK293 cells transfected with the WT NKCC1 transporter or NKCC1 mutated at different residues. The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC1-mediated Cl– influx. (C) Quantification of the mouse NKCC1 inhibitory activity using the Cl– influx fluorescence assay in HEK293 cells. A fluorescence signal decrease, corresponding to a decrease in NKCC1 transporter activity, was observed for all the cells transfected with NKCC1 mutants. Data are normalized and the average of the last 10 s of kinetics is plotted (ΔF/F0). Data are presented as a percentage of the WT. Data represent mean ± SEM from 3 to 4 independent experiments (Kruskal–Wallis one way ANOVA, H = 216, DF = 6, followed by Dunn’s post hoc test on multiple comparisons, *** P = 0.0002, **** P < 0.0001).

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In our in vitro cellular assay, all mutations lead to a decrease in ion transport function, when compared to the wild-type (WT) mouse NKCC1 (Figure 7C). In detail, the selected mutations were Ala490Trp (equivalent to human NKCC1 Ala497Trp at TM 6, alignment shown in Figure S9). This mutation introduces a bulky side chain that disrupts the formation of the Ser 679 – Pro 496 interaction at the TM 6 – TM 10 interface at the outer vestibule. Ala490Trp mutation led to reduced transport by a factor of 1.7 when compared to the WT NKCC1. We infer that by affecting TM 10's motion, the NKCC1’s IO state is destabilized, therefore disrupting ion transport. Additionally, the mutations Leu664Ala, Asn665Ala and Ala668Trp (equivalent to human Leu671Ala, Asn672Ala and Ala675Trp, see Figure S9 - all residues located in the extracellular tip of TM 10) also led to a reduction in ion transport by a factor of 1.4, 1.3, and 1.6, respectively, when compared to the wild-type NKCC1 (Figure 7C). In this context, the Leu664Ala and Asn665Ala mutations on TM 10 remove the side chain that interacts with the Asn672 and Ile493 residues on TM 10 and TM 6, respectively. The mutation Ala675Trp introduces a bulky side chain that disrupts the formation of the Pro 676 – Ala 492 interaction, weakening the TM 10 mobility (Figure 7A).
Overall, our simulations and mutagenesis data support a rocking-bundle mechanism for NKCC1 conformational transitions, which critically relies on the TM 4 and TM 9’s angular motion and dynamic contacts of residues at the TM 6 – TM 10 interface, with TM 10 that seems to also modulate the access of the solvent to the outer vestibule.

Free Energy Simulations Characterize the Previously Elusive NKCC1’s Occluded State

Then, we analyzed the free energy surface (FES) for IO ↔ OO conformational transitions of NKCC1 as revealed by the OPES Explore algorithm and CVs used to enhance the sampling of the complex dynamical transitions between open conformations of the inner and outer vestibules of the transporter (see the paragraph above and Methods section). Our simulations revealed 4 distinct free energy minima that correspond to conformational states of NKCC1. Notably, the IO state is located in the deepest minimum. We found that the IO state basin was centered on a value of −1.6 in the CV dimension (Figures 8 and S6). Further equilibrium MD starting from snapshots extracted from this basin showed a stable IO model (Figure S7A). On the other hand, the OO state was distributed into a shallow area located at ∼2.6 in the CV dimension of the FES. However, we found this area to cover different hydration states of the transporter (OOw and OOd in Figure 8). That is, both OOw and OOd are stable OO state conformations that differ in the extent of their water hydration (Figure S7B). Indeed, the outer vestibule in the OOd minimum has a coordination number of waters of ∼10.5, while the outer vestibule in OOw showed a coordination number of ∼20.5.

Figure 8

Figure 8. Free energy surface identifies relevant NKCC1 conformations for the inward open ↔ outward open transition. (A) Representation of the Free Energy Surface of the conformational transition between human NKCC1 IO and OO states computed by OPES Explore over the DeepLDA collective variable and the outer vestibule water coordination collective variable. Energetical basins are highlighted with a schematic representation of the conformation they identify. These are, namely: the IO state, the occluded state (labeled Occ), the OOd state (outward open “dry” – lower outer vestibule hydration) and OOw (outward open “wet” – higher outer vestibule hydration. (B) Higher magnification of the hexagon in A representing the main gating interactions that occlude the outer vestibule and block solvent access to the ionic binding sites. Relevant residues are shown as sticks with their atomic surfaces pictured in red (oxygen), blue (nitrogen), gray (carbon) and white (hydrogen). (C) Higher magnification of the hexagon in A representing of the main gating interactions that occlude the inner vestibule and block solvent access to the ionic binding sites. Relevant residues are shown as sticks with their atomic surfaces pictured in red (oxygen), blue (nitrogen), yellow (sulfur), gray (carbon) and white (hydrogen).

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Importantly, our exploration of FES also captured NKCC1 in its occluded state (Occ, Figure 8A). Notably, such NKCC1 occluded state had been only hypothesized to exist, (27) as it has never been structurally determined, likely due to its transitory nature. This state was located at ∼0 in the CV space with an outer vestibule hydration coordination number of 4.8. Notably, such an occluded state emerged naturally from our deep-learning-guided enhanced sampling simulations trained solely on the IO and OO state conformations.
In our simulations, the occluded state depicts NKCC1 with its ion binding sites inaccessible to the solvent, from either side of the membrane (Occ in Figure 8A), and it is characterized by having both outer and inner vestibules occluded. The outer vestibule has an extensive intermolecular interaction hub. This was built up by Arg 307, which formed a salt bridge with Glu 389, and cation-π interactions with Phe 590 (Figure 8B). Additionally, the interactions between Asn 672-Ile 493, Pro 676-Ala 492 and Ser 679-Pro 496 were fully formed in the NKCC1 occluded state, thus occluding access of the extracellular solvent to the outer vestibule. On the other side, the inner vestibule was occluded to the intracellular solvent due to several other interactions among residues. Those interactions were mostly conserved also in the OO state of the cryo-EM structure. (15) Primarily, these interface residues’ interactions are formed after the shifting of Met 428 and the consequent occlusion by intracellular loop 1. Interestingly, the salt bridge between Asp 510 and Lys 624, formed in the OO state where it occludes the inner vestibule (Figure 8C), is not formed in the occluded state. Notably, the occluded state was maintained for over 100 ns in our equilibrium MD simulations, which started from configurations of the Occ state visited during the enhanced sampling trajectories (Figure S7C), with an RMSD of 1.00 ± 0.11 Å. In these simulations, all ion binding sites remained inaccessible to the solvent from either side of the membrane, as shown in the occluded basin pore profile in Figure 9B. Notably, this differs greatly from the IO and OO state equilibrium MD pore profiles (Figure 9A,C, respectively), confirming that such a transitory state is structurally distinct from the IO and OO states. Ultimately, the occluded state is therefore the only transitory conformation that ensures solvent inaccessibility to the ion binding sites from both the intra and extracellular vestibules.

Figure 9

Figure 9. Pore profile confirms distinct binding-site accessibility of NKCC1 states along the inward open ↔ outward open transition. (A–C) Pore profile of the IO state (A), the occluded state (B), and OO state (C), schematically represented at the bottom. These profiles were obtained by calculating the radius of the largest sphere along the Z-axis of the ion translocation cavity, and then plotting the pore profile of several snapshots from their corresponding equilibrium MD simulations, computed by the software HOLE. Pore profiles were mirrored around radius 0 for visual clarity. The blue lines represent the outer vestibule, and the orange lines represent the inner vestibule of NKCC1.

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In our FES (Figure 8A), the IO state transited to the occluded state, with a barrier of ∼5.5 kcal/mol. From this transitory occluded state, there is another barrier of ∼5.5 kcal/mol to reach the OOw state. Interestingly, the reverse transition appears less energetically costly, as the OOw state transited to the occluded state with a barrier of ∼3.5 kcal/mol and then followed by a ∼0.5 kcal/mol barrier to reach the IO state. Therefore, the IO state was ∼7 kcal/mol more stable than the OOw state. Taken together, our simulations depict IO ↔ OO transitions passing though the occluded state, further corroborating the rocking-bundle mechanism for NKCC1 function.
Then, we determined the free energy for ion binding to the NKCC1 OO state. According to previously published kinetic data, (28) ions bind to NKCC1 from the extracellular side of the membrane in the order Na+, Cl–, K+, Cl–. We first simulated Na+ binding to the unloaded OO state (Figure S11A), and found a ΔGbind ≈ −7.5 kcal/mol, with a binding barrier of ∼5.0 kcal/mol. We then evaluated binding of the first Cl– ion to a Na+-bound OO state (Figure S11B), which returned a ΔGbind ≈ 1.0 kcal/mol, with an energy barrier of ∼4.0 kcal/mol. Next, we evaluated K+ binding to a Cl–-Na+-bound OO state (Figure S11C), and we obtained a ΔGbind ≈ of 1.0 kcal/mol, with a barrier of ∼4.5 kcal/mol. After these ion binding simulations, we ran additional ∼100 ns of equilibrium MD of each system and observed that all these partially/fully loaded states were stable, i.e., all ions remained stably coordinated to their binding site throughout the simulations time. Interestingly, within the first 5 ns of the K+-Cl–-Na+-bound OO NKCC1 state, we observed the spontaneous binding of the second Cl– ion to its binding site, leading to the fully loaded OO state of NKCC1, which was stably maintained for over 1 μs.
We analyzed the ion binding sites and ion coordination to inspect possible effects of the conformational transition OO → IO on those ion binding sites. First, we observed that ion loading does not affect the overall structure of NKCC1 (RMSD between the loaded and unloaded states: IO = 1.71 Å, occluded = 1.97 Å, OO = 1.63 Å) and that the fully loaded occluded → IO state conformational transition is characterized by the same angular motion of TM 4 and TM 9 (Figure S10). In addition, we noticed that all ions remained bound to their respective sites throughout the conformational transition OO → IO, although minor changes were detected. In detail, the Na+ binding site is composed by residues: Leu 297, Trp 300, Ala 610, Ser 613, and Ser 614. Na+ maintains its interactions with most of its coordination sphere in the fully loaded OO, occluded and IO states (Figure S12A). The only state-dependent interactions are between Na+ and Ala 610. This distance started at an average length of 2.7 ± 0.7 Å in the OO state (i.e., tightly bound) and increased in both occluded and IO states to 4.4 ± 0.5 and 4.6 ± 0.4 Å, respectively (i.e., loosely bound). The interactions of the first Cl– to its binding site (closest to the intracellular side), are with residues: Gly 500, Ile 501, Leu 502, and Tyr 686. These remained unchanged between the fully loaded OO, occluded and IO states (Figure S12B). Most of K+ interactions with its binding site, which is composed by residues: Asn 298, Ile 299, Tyr 383, Pro 496, and Thr 499, are stably maintained through NKCC1’s conformational transition (Figure S12C). The exception is the interaction between the bound K+ and residue Asn 298, which changes during the conformational transition. This K+ is initially tightly bound in the OO state (average distance to Asn 298 = 2.9 ± 0.3 Å) to become loosely bound in the Occ and IO states (average distance to Asn 298 = 4.1 ± 0.8 and 4.4 ± 0.7 Å, respectively). The interaction with Thr 499 also changes during the conformational transition, although this occurs at a different stage of the process. In both OO and Occ states, K+ remains tightly bound (average distance to Thr 499 = 2.7 ± 0.2 and 2.8 ± 0.2 Å, respectively) but becomes loosely bound in the IO state (average distance to Thr 499 = 4.3 ± 1.6 Å). On the other hand, the second Cl– has a distinctive binding mode to its binding site (closest to the extracellular side), which is composed by residues: Val 302 and Met 303. In the OO state, it is loosely bound to both Val 302 and Met 303 (average distance to each residue being 4.5 ± 1.7 and 5.2 ± 1.8 Å, respectively). Whereas it is tightly bound in the occluded state (average distance to each residue being 2.4 ± 0.2 and 2.6 ± 0.2 Å, respectively) and IO state (average distance to each residue being 2.5 ± 0.3 and 2.7 ± 0.3 Å, respectively). These results further support that the second Cl– has the propensity of spontaneously binding to the OO state.

NKCC1 Permeability Allows Water Transportation

To better understand the capabilities to transport water of NKCC1, we modeled two new additional conformational states. These were IO and OO, which we loaded with two Cl–, Na+ ions, and K+ ions in their respective binding sites. These two fully loaded models allowed us to evaluate water permeability and water transport in NKCC1 in the presence of ions (as opposed to the previous models that were studied in the absence of ions). Notably, we observed an average of 13 water molecules trapped in the ion binding cavity through 100 ns of equilibrium MD simulations of the occluded state without ions bound. This value would then represent an estimation of the maximum amount of water molecules transported per alternating access cycle. Interestingly, our simulations also showed that NKCC1 adopts water-permeable states in the OO state (Figure 10A), preferentially when unloaded (38.2% of the trajectory, Figure 10B), while it does so only to a minor extent in the OO state with 4 ions bound (1.1%, Figure 10C). Finally, NKCC1 is not permeable at all in the loaded and unloaded IO and Occluded states.

Figure 10

Figure 10. NKCC1’s outward open conformations are permeable to water. (A) Representation of NKCC1 (gray cartoon) embedded in the membrane (black horizontal bars) in a water-permeable state. Water molecules are shown as red and white lines with a red atomic surface representation. A chain of water molecules whose oxygen atoms are within 4.0 Å of each other connecting the extracellular and intracellular solvent is present. (B, C) Schematic representation of both states that present permeability to water (outward open without ions bound, B, or fully loaded, C), and their respective plot, which tracks the appearance of permeable states during each state’s equilibrium MD simulation. Vertical red bars represent snapshots from the respective simulation where a chain of water molecules whose oxygen atoms are within 4.0 Å of each other connecting the extracellular and intracellular solvent through NKCC1 is observed.

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Water-permeable states of NKCC1 have an average lifetime of 0.5 ± 0.5 ns, with a median lifetime of 0.2 ns and a maximum lifetime of 5.0 ns in the unloaded OO conformation. On the other hand, water-permeable states in the OO fully loaded state have an average lifetime of 0.3 ± 0.2 ns, with a median of 0.2 ns and a maximum lifetime of 1.0 ns. This finding is in line with our mechanistic insights provided by the enhanced sampling simulations, which showed that in the IO states TM 10 modulates the hydration of the outer vestibule. Ultimately, when NKCC1 adopts a permeable state, water molecules diffuse across the membrane. During the 1 μs-long MD simulations of the OO state, we tracked 517 complete water molecule efflux events and 497 influx events (Figure 11). It is to be noted that these were pure diffusion events, as our MD simulations did not include any concentration gradient across the membrane. With this premise, we estimated the average water diffusion rate as 1.8 ± 2.5 and 2.0 ± 4.2 ns, in the outward and inward direction, respectively.

Figure 11

Figure 11. NKCC1 passively transports water. Histogram of all transport events detected in the state equilibrium MD simulation of NKCC1 outward open conformation with no ions bound, organized by the length of each transport event. Bars show the frequency of efflux/influx (orange/green) events per transport event duration. Vertical continuous lines show the mean, and discontinuous lines show the standard deviation (efflux, orange; influx, green). Some longer transport events were excluded from the histogram for clarity.

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Discussion

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The CCC NKCC1 plays a crucial role in cellular osmolarity by regulating ionic balance and water flux, (29) and it is currently targeted to treat a variety of related imbalance diseases, e.g. brain disorders including neurodevelopmental, neurodegenerative, neurological, disorders, hydrocephalus, as well as cancer. (3−7) Motivated by the recent structural data on NKCC1 in different conformations─IO and OO states─we built and simulated atomistic models of human NKCC1, where the transporter is embedded in the membrane. We used these models to run multiple μs-long MD equilibrium and enhanced sampling simulations of those key IO and OO states and capture the exact protein dynamics for IO ↔ OO transitions.
Notably, our investigation led to a grand total of 16 conformational transitions observed using the OPES algorithm for enhanced sampling simulations. (19) These simulations revealed a rocking-bundle mechanism for conformational transitions for the unloaded NKCC1 protein. Specifically, such a rocking-bundle mechanism shows alternate access to the NKCC1 ion binding sites through two concerted motions. First, helices TM 4 and TM 9, associated with each other by hydrophobic interactions, go through an angular motion of ∼14° between the IO and OO states. This is then coupled by the motion of TM 10, linked by a short extracellular loop to TM 9. The latter alternatively affects the bending or straightening of TM 10, thus blocking/allowing solvent access to the ion binding sites from the extracellular side. Such motions were consistently observed in all our IO ↔ OO state transitions (see Movie S1). We also observed these motions in our fully loaded NKCC1 model, suggesting that NKCC1 undergoes a rocking-bundle mechanism with and without bound ions. Remarkably, NKCC1’s TM 4, TM 9, and TM 10 behaviors are conserved in the LeuT-fold sodium-benzylhydantoin transporter Mhp1 from Mycobacterium liquefaciens, (30) where the same TM helices may therefore carry out an analogous role for function.
Importantly, from our enhanced sampling simulations, we also identified the so-called “occluded state” for NKCC1, where the ion binding sites are inaccessible from both sides of the transporter (i.e., both the inner and outer vestibules are closed). Notably, the occluded state was only hypothesized to exist based on previous structural studies, (27) although it has never been experimentally observed in any of the CCCs. On the other hand, in our simulations, we like to emphasize that the deep-learning-guided sampling of NKCC1 conformations detected such an occluded state without any previous knowledge of its existence. In other words, this previously uncharacterized occluded conformational state was not used to generate our data set and identify proper CVs for deep-learning enhanced sampling simulations, which therefore have unexpectedly revealed such a minimum on the FES, with no external solicitation. In addition, the OO state shows two distinct conformations, distributed into a shallow area located at ∼2.6 in the FES. These conformations (OOw and OOd in Figure 8) show a difference in the extent of their water hydration (Figure S7B) at the vestibules due to the modulatory motion of TM 10. Interestingly, the hydration level of the outer vestibule in the OO state was significantly affected by the Arg 307–Glu 389 salt bridge (Figure S8). It is also worth noting that the drug bumetanide is bound to the outer vestibule in all the available cryo-EM structures of NKCC1 in the OO state. (12,15) Ligand binding at the outer vestibule hampers the formation of the Arg 307-Glu 389 salt bridge, which is, therefore, proven to be crucial for the rocking-bundle mechanism in NKCC1.
Overall, the IO state is found to be the lowest energy basin of the FES. From the IO state, the transporter transits to the OO state, passing through the occluded state. Our semiquantitative estimation of the energetic cost for the IO → OO transition is ∼10.5 kcal/mol, whereas the estimated cost for the OO → IO transition is ∼3.5 kcal/mol. Interestingly, these energetic estimates would also explain why all of the so-far resolved NKCC1 apo structures have been captured in the lowest free energy IO state minimum.
Another interesting observation is related to the possibility that NKCC1 can transport water molecules. Here, we have observed and quantified the ability of water to passively permeate through NKCC1. Using additional simulations with 4 ions bound to the IO and OO states, we could compare the presence of water molecules in different models and observe that NKCC1 indeed adopts water-permeable states exclusively in the unloaded OO state (38.2% of the trajectory, Figure 10B). We found that NKCC1’s water-permeable states have an average lifetime of 0.5 ± 0.5 ns, with water molecules that can diffuse across the membrane. In our MD simulations of the OO state, we tracked 517 complete efflux events and 497 influx events (Figure 11), which occur through diffusion (see Movie S2), as our MD simulations do not imply concentration gradient across the membrane. With this premise, we estimated the average water diffusion rate, which is 1.8 ± 2.5 and 2.0 ± 4.2 ns, in the outward and inward direction, respectively. Water transport may also take place via the alternating access cycle. During the simulation of the occluded state without bound ions, we observed about 13 water molecules trapped in the ion binding region. The latter can be therefore considered an estimation of the maximum amount of water molecules transported per alternating access cycle. Interestingly, these semiquantitative observation enrich the current experimental evidence for water transport by NKCC1 (31,32) and its functional implications in the context of CSF accumulation recently demonstrated. (33,34) Indeed, water may cross the membrane via the formation of permeable states, as has been reported for a wide range of transporters, including Na+/glucose transporter (SGLT), glutamate transporter (Gltph), glycerol-3-phosphate transporter (GlpT), sodium-benzylhydantoin transporter (Mhp1), and the maltose transporter, (35) and more recently also shown for the sodium/proton antiporter PaNhaP. (36)
One more mechanistic feature from our simulations is the interhelical interface intrinsically associated with NKCC1’s transport capabilities. This interface is formed by the extracellular tip of helices TM 10 and TM 6. In particular, the interatomic contacts at the TM 10–TM 6 interface are maintained in the IO state (Figure 5), allowing sealing of the outer vestibule in the IO state. This interaction will then be broken to transit to the OO state, aided by the specific rocking-bundle mechanism and the associated helice motions (TM 4 and TM 9, Figure 2). To prove the relevance of such a critical protein interface, we also mutated a few residues located in it. From mouse NKCC1, we inserted the following mutations: Ala490Trp in TM 6, and Leu664Ala, Asn665Ala, and Ala668Trp in TM 10 (equivalent to human NKCC1 Ala497Trp, Leu671Ala, Asn672Ala, and Ala675Trp, respectively). All of these mutations led to a significant reduction in ion transport (Figure 6). This is also in line with previous cross-linking experiments, which highlighted the significant movement of TM 10 during NKCC1 ion transport. (37) This further validates the key modulatory motions reported here for TM 10. Notably, TM 4’s functional importance is supported by mutational data showing that the Arg410Gln mutation─located at the extracellular extreme of TM 4─leads to a loss of function. (38) Our computational evidence is therefore well supported by our and literature’s mutagenesis data.
Additionally, we characterized the energy profile of the binding of ions to the OO state of NKCC1. Crucially, we found that the Na+ dissociation energy in the OO state is 12.5 kcal/mol, while its dissociation energy in the IO state has been previously reported to be 6.9 ± 0.8 kcal/mol. (39) We note that the free energy calculations for ion release that pertain to the IO state (39) were performed via well-tempered metadynamics, while ours pertain to the OO state for ion binding and unbinding, computed using OPES Explore simulations. While qualitative, these results hint at anyhow to a higher binding affinity of Na+ to the OO state than to the IO state. Notably, this is congruent with NKCC1’s use of a Na+ electrochemical gradient to import Cl– and its overall functional cycle─where ions bind to the OO state from the extracellular side of the membrane and are then released from the IO state into the intracellular side of the membrane.

Extension of Our Mechanistic Implications to Na+-Dependent CCCs

Intriguingly, the results on the functional relevance of TM 4 and TM 9, along with TM 6 and TM 10, motivated our analysis of additional mutations of CCCs in this region, often linked to the insurgence of pathologies. (40) In doing so, we found a total of 38 mutations (including the new 4 mutations reported here), which are all located on the functionally relevant helices identified in our work, namely, TM 4, TM 6, TM 9, and TM 10, highlighted in red in Figure S9. These four TM helices present an extremely high level of conservation (considering either identity and similarity) in Na+-dependent CCCs─between 80 and 100% when compared to human NKCC1, as shown in Figure S9. On the other hand, these functionally relevant TM helices present a much lower degree of conservation in Na+-independent CCCs, with values ranging from 40 to 73% when compared to human NKCC1. Therefore, the rocking-bundle mechanism seems to operate only for Na+-dependent CCCs, to which NKCC1 belongs. Na+-independent CCCs (KCCs) may operate through a different alternate access mechanism, where transitions seem driven by different TM helices, like TM 3 and TM 8 in the human KCC1. (41) In particular, we could only find three mutations in our region of interest in Na+-independent CCCs that lead to transporter dysfunction, and all these are in TM 6. However, these three mutations are reported to have functional effects that seem unrelated to the rocking-bundle alternating access mechanism. Specifically, the human KCC2’s Leu426Pro mutation leads to complete loss of protein function, with reduced expression and glycosylation. (42) The human KCC2’s Met438Val mutation alters the Cl2 binding site (referenced on the cited article as Met415Val because it is mutated in KCC2b) and therefore leads to impaired Cl– extrusion. (42) Finally, the Phe493CysfsX48 mutation in human KCC3 is associated with a frameshift mutation, which generally carries serious pathogenic consequences, like in this case with agenesis of the corpus callosum. (43)

Conclusions

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In summary, our computational simulations and free energy calculations, coupled to mutagenesis experiments, show that NKCC1 operates through the rocking-bundle mechanism, transiting from the unloaded IO to the OO states and vice versa. We also found that the OO states are permeable to water, which can freely go through NKCC1 across the membrane. Importantly, we found that most functionally relevant TM helices involved in such a mechanism are highly conserved in Na+-dependent CCCs. This overall evidence and critical mechanistic implications could open new strategies for NKCC1 function modulation and novel modes of drug action.

Methods

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Equilibrium MD

The IO state model was constructed based on the cryo-EM structure of apo human NKCC1, (13) using the TM domain of one monomer (comprising residues 288–753). The OO state model was built from the last snapshot from previous simulations, (15) from which we removed bumetanide and bound ions and allowed water to flood the space previously occupied by the removed elements. For both states, the NKCC1 monomer was embedded in a POPC bilayer. PropKa 3.0 (44) was used to determine the ionization state of titratable residues, assuming pH 7─all calculated pKa values were conducive to the standard ionization state at pH 7. The simulation box included NKCC1 accompanied by 221/308 POPC molecules, 59/63 Cl–, 29/31 Na+, and 31/33 K+ ions (in the bulk solution, corresponding to ∼150 mM concentration), and ∼19,300/22,000 water molecules. In total, ∼ 95,000/114,000 atoms and a simulation cell of 87 Å × 96 Å × 110 and 100 Å × 115 Å × 97 Å dimensions, for the IO and OO states, respectively. Initial configurations of each model were assembled using Packmol-Memgen, (45) part of the AmberTools software package.
Equilibrium MD simulations were performed using the GPU version of the PMEMD code (46) of the AMBER package. (47) The protein was modeled using the ff14SB force field, (48) Lipid17 for the POPC bilayer, (49) TIP3P for water (50) and for the ions. (51) Periodic boundary conditions were employed, using the particle mesh Ewald method to calculate the long-range electrostatics. (52) The real part of the electrostatic and van der Waals interactions were computed with a 10 Å cutoff. The SHAKE algorithm (53) was used to constrain bonds involving hydrogen atoms, allowing an integration time step of 2 fs. Simulations were performed at a constant temperature (310 K) and pressure (1 bar). The POPC bilayer and water solvent were allowed to equilibrate around the protein during 200 ns of the MD simulation. After energy minimization, the system was gradually heated to 310 K, with the protein backbone atoms maintained close to their cryo-EM position by applying a harmonic restraint. Then, 1 μs of production equilibrium MD was performed for each IO and OO state NKCC1.

Conformational CV Design

To guide the exploration of such complex systems and transitions, we applied the OPES Explore algorithm, which requires the use of effective CVs to accelerate a meaningful sampling of the free energy surface. By combining OPES Explore with the DeepLDA CV that we developed, our simulations could capture the complex dynamical transitions between open conformations of the inner and outer vestibules of the transporter. Using the IO and OO state equilibrium MD simulations, we selected all Cα–Cα pairs from all 12 TM helices, totaling to ∼75,000 Cα–Cα distances. These were then subsequently filtered, eliminating intra-TM pairs and keeping pairs only from adjacent helices. Then, we kept Cα–Cα pairs whose average distance was deemed a contact (<10 Å) in the IO state and not a contact in the OO state (>10 Å) and vice versa. Finally, we retained Cα–Cα distances that were significantly different between the IO and OO states, by eliminating those whose average was within two standard deviations between states. This resulted in a carefully curated selection of 90 Cα–Cα distances (see also Figure S2). From each 1 μs-long equilibrium MD, 50,000 data points per distance and per state were then fed to DeepLDA. (18) This produced a deep-learning CV where the IO state was defined as −2.6 and the OO state was defined as 2.6.

Water CV Design

The conformational CV was sufficient for enhanced sampling simulations to transit between the IO and the OO states, but we observed that these simulations were getting trapped in a state with the outer vestibule open and highly solvated. To aid water flushing from the outer vestibule and reduce steric hindrance of water impeding structural rearrangements, we included a second water-focused CV.
This CV consisted of the coordination number between a virtual atom placed in the center of the outer vestibule (Figure S3) and the oxygen atoms of water molecules. (54,55) The coordination was calculated using a switching function (see Formula 1) with the following parameters: r0 = 8.0 Å, d0 = 0, n = 2, and m = 8. This CV characterizes the water content of the outer vestibule, and when its fluctuations were enhanced through a bias potential, we were able to obtain 16 IO ↔ OO state transitions:
s(r)=1−(r−d0r0)n1−(r−d0r0)m
(1)
Switching function for the outer vestibule virtual atom–water coordination number.

OPES Explore

We performed bidimensional OPES Explore (19) on the DeepLDA CV and outer vestibule water coordination CV through PLUMED 2.8. (56) We set an initial barrier value of 20 kcal/mol, a kernel deposition rate of 500 steps, and a 310 K temperature. The initial sigma was set to 0.1 and 0.3, and the minimum sigma was set to 0.05 and 0.15, for the conformational and water CV, respectively.

Fully Loaded NKCC1 Conformations and Ion Binding Simulations

Fully loaded IO human NKCC1 was obtained by placing ions in their binding sites in accordance to their position in the zebrafish NKCC1 structure, (11) except for the Na+ cation, which was placed in the center of mass of the coordinating atoms of the known Na2 binding site also identified in human NKCC1. (13) We obtained a stable fully loaded IO NKCC1 conformation, in agreement with previous computational studies. (39) This conformational state was then simulated for 1 μs.
Fully loaded occluded human NKCC1 was obtained by applying distance restrictions to each ion and all coordinating atoms, from each ion’s respective binding site. The application of these restraints, for 100 ns, led to a NKCC1 state with all four ions bound and inaccessible to the solvent from both extracellular and intracellular sides of the membrane─a fully loaded occluded state. After restrictions were lifted, this state was maintained for 120 ns of equilibrium MD, after which NKCC1 was transitioned into a fully loaded IO state.
Fully loaded OO human NKCC1 was obtained by starting from previous simulations, (15) where NKCC1 was bound to bCl– (Cl– anion closest to the intracellular side), K+, and the inhibitor bumetanide. Bumetanide was removed, K+ was exchanged for Na+ from the solvent, and the outer vestibule was allowed to be filled with water. After equilibration, we ran OPES Explore simulation biasing two CVs. The first CV is the distance between the center of mass of the Na+ site coordinating atoms and the Na+ cation, currently bound to the K+ site. The second CV was the coordination number between the Na+ cation and the coordinating atoms of the Na+ site. We then selected a snapshot where the Na+ cation was within its binding site and ran for 100 ns of equilibrium MD. We observed that bCl– and Na+ both stayed in their respective binding site through the 100 ns of simulation. We then selected the closest K+ to the outer vestibule from the solvent and ran a second OPES Explore over two similar CVs, but instead considering the K+ cation and the K+ binding-site coordinating atoms. Then, a snapshot where K+ was bound to its site was selected and used for an equilibrium MD. After an initial run of 100 ns, we observed that not only bCl–, Na+, and K+ remained in their respective binding sites but spontaneous binding of tCl– to the top Cl– binding site was observed very soon after the equilibrium MD started. Given that this last simulation was of NKCC1 in an OO fully loaded state, we extended it to 1 μs.
Na+ and Cl– binding calculations were performed as described in the previous paragraph. OPES Explore simulations of Na+ binding started from a snapshot from our unloaded OO state NKCC1 equilibrium MD simulation. A snapshot where a Na+ ion was nearby the outer vestibule was selected and bias was applied to the distance between the ion and the center of mass of the coordinating atoms of the Na+ binding site and to the coordination number between Na+ and the coordinating atoms. OPES Explore simulations of Cl– binding started from the previously equilibrated MD simulation of Na+/Cl–-bound OO state NKCC1, using the same set of CVs, considering Cl– and its respective binding site. Ion binding order to the OO state of NKCC1 (Na+, Cl–, K+, and Cl–) was determined from experimental evidence. (28)
For the OPES Explore simulations of NKCC1 loading, the barrier was set to 5 kcal/mol, a kernel deposition rate of 500 steps, and a 310 K temperature. Both initial and minimum sigmas were set to adaptive for both CVs. The coordination for all ions was calculated using a switching function (see Formula 1) with the following parameters: r0 = 2.35 Å, d0 = 0, n = 2, and m = 8.

Water Permeability and Transport

Water permeability was defined as the presence of a network of water molecules, connected by a distance of at most 4.00 Å between oxygen atoms from water molecules that encompassed the whole ion translocation pathway, connecting the extracellular and intracellular solvent (Figure 10).
To determine water transport in our simulations, we tracked each individual water molecule. An efflux event was determined to have happened when a water molecule crossed the inner membrane leaflet plane, the plane of the membrane bilayer, and then the outer membrane leaflet plane in this sequence specifically. An influx event was determined to have happened because a water molecule went through these planes in the reverse direction. Analysis was mostly performed with the MD analysis package. (57)

Experimental Section

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Generation of NKCC1 Mutants and Cl– Influx Assay

Generation of Mutants

Mutants of residues located in TM 6 and TM 10 in mouse NKCC1 [Ala490 (TM 6), Leu664 (TM 10), Asn665 (TM 10), Ala668 (TM 10), and mutating to Ala or Trp] were designed based on MD simulations. NKCC1 mutants (Ala490Trp, Leu664Ala, Asn665Ala, and Ala668Trp) were designed starting from the full-length mouse NKCC1-WT protein sequence cloned in the vector pRK5 (obtained from the Medical Research Council and the University of Dundee). The mutants were prepared by GenScript. For each mutant, the lyophilized DNA was resuspended and used to transform Escherichia coli TOP10 competent cells, and a maxi prep was performed to purify the DNA of each mutant. The sequences were then confirmed by Sanger sequencing.

Cl Influx Assay

HEK293F cells were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained at 37 °C in a 5% CO2 humidified atmosphere. To assess WT and mutant NKCC1 activity, 3 million HEK cells were plated in a 10 cm cell-culture dish and transfected with a transfection mixture comprising 5 mL of DMEM, 4 mL of Opti-MEM, 8 μg of DNA plasmid (pRK5 vector) coding for NKCC1-WT, NKCC1-A490W, NKCC1-L664A, NKCC1-N665A, NKCC1-A668W, or mock control (empty vector), together with 8 μg of a plasmid coding for the Cl-sensitive variant of the mbYFPQS (Addgene plasmid #80742), and 32 μL of Lipofectamin 2000. After 4 h, the cells were collected and plated in 96-well black-walled, clear-bottomed plates at a density of 250,000 cells/well. After 48 h, cells were used for the Cl influx assay. All reagents were purchased from Life Technologies unless otherwise specified. The Cl influx assay was performed in transfected cells treated with DMSO in 200 μL/well of a Cl-free-hypotonic solution (67.5 mM Na Gluconate, 2.5 mM K Gluconate, 15 mM HEPES pH 7.4, 5 mM Glucose, 1 mM Na2HPO4, 1 mM NaH2PO4, 1 mM MgSO4, and 1 mM CaSO4). After 30 min of incubation, plates were loaded into a multiplate reader (Tecan Spark) equipped with an automatic liquid injector system, and fluorescence of Cl-sensitive mbYFPQS was recorded with excitation at 485 nm and emission at 535 nm. For each well, fluorescence was first recorded for 20 s of baseline and for 60 s after delivery of a NaCl-concentrated solution (74 mM final concentration in the assay well). Fluorescence of Cl-sensitive mbYFPQS is inversely correlated to the intracellular Cl– concentration, therefore, Cl influx into the cells determined a decrease of mbYFPQS fluorescence. To quantify the average effects as represented by the bar plots, we expressed the decrease in fluorescence upon NaCl application as the average of the last 10 s of ΔF/F0 normalized traces. Moreover, for each experiment, to account for the contribution of Cl– changes that were dependent on transporters/exchangers other than NKCC1, we subtracted the value of the last 10 s of ΔF/F0 normalized traces obtained from mock-transfected control cells from the respective ΔF/F0 values obtained from the cells transfected with WT or mutated NKCC1s. We then presented in the figure all the data as a percentage of the fluorescence decrease vs the value of the WT NKCC1-transfected cells.

Data Availability

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Trajectories are available upon request, while representative structures from all states (inward open, occluded and outward open in both fully loaded and ion-free states) are available in MJRM’s github repository (https://github.com/themanuelr/NKCC1_rep_struc).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c10258.

  • Alternating access mechanisms, collective variable design, and relevant TM helices sequence alignment, and further analysis of the equilibrium and enhanced sampling MD simulations (state stability, salt-bridge formation and water content, and others) (PDF)

  • Angular motion for the rocking-bundle mechanism (MP4)

  • Presence of water-permeable states (MP4)

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Author Information

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  • Corresponding Authors
    • Michele Parrinello - Laboratory of Atomistic Simulations, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy;  Email: [email protected]
    • Laura Cancedda - Laboratory of Brain Development and Disease, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy;  Email: [email protected]
    • Marco De Vivo - Laboratory of Molecular Modelling & Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy;  Orcidhttps://orcid.org/0000-0003-4022-5661;  Email: [email protected]
  • Authors
    • Manuel José Ruiz Munevar - Laboratory of Molecular Modelling & Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    • Valerio Rizzi - Biomolecular & Pharmaceutical Modelling Group, Université de Genève, Rue Michel-Servet 1, Geneva CH-1211 4, Switzerland;  Orcidhttps://orcid.org/0000-0001-5126-8996
    • Corinne Portioli - Laboratory of Nanotechnology for Precision Medicine, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy;  Laboratory of Brain Development and Disease, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    • Pietro Vidossich - Laboratory of Molecular Modelling & Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, Genoa 16163, Italy
    • Erhu Cao - Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, United States
  • Notes
    The authors declare the following competing financial interest(s): L.C. is named as co-inventor on the following granted patents: US 9,822,368, EP 3083959, and JP 6490077; M.D.V., and L.C. are named as co-inventors on patent application IT 102019000004929. L.C. and M.D.V. are scientific founders and act as consultants of IAMA Therapeutics.

Acknowledgments

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We thank Narjes Ansari for useful scientific discussions. L.C. thanks Telethon for financial support (Grant TCP15021). L.C. thanks the European Research Council (ERC) for partial funding (Grant agreement no. 725563). C.P. thanks the Marie Skłodowska-Curie Action for financial support (Grant agreement no. 843239), under the European Union’s Horizon 2020 research and innovation programme. E.C. thanks the NIH for financial support (Grant DK128592).

References

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This article references 57 other publications.

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    The choroid plexus epithelium (CPE) secretes higher vols. of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood-CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little exptl. evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is assocd. with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4-NF-κB signaling or the SPAK-NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.
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    Bumetanide (BTN or BUM) is a FDA-approved potent loop diuretic (LD) that acts by antagonizing sodium-potassium-chloride (Na-K-Cl) cotransporters, NKCC1 (SLc12a2) and NKCC2. While NKCC1 is expressed both in the CNS and in systemic organs, NKCC2 is kidney-specific. The off-label use of BTN to modulate neuronal transmembrane Cl(-) gradients by blocking NKCC1 in the CNS has now been tested as an anti-seizure agent and as an intervention for neurological disorders in pre-clinical studies with varying results. BTN safety and efficacy for its off-label use has also been tested in several clinical trials for neonates, children, adolescents, and adults. It failed to meet efficacy criteria for hypoxic-ischemic encephalopathy (HIE) neonatal seizures. In contrast, positive outcomes in temporal lobe epilepsy (TLE), autism, and schizophrenia trials have been attributed to BTN in studies evaluating its off-label use. NKCC1 is an electroneutral neuronal Cl(-) importer and the dominance of NKCC1 function has been proposed as the common pathology for HIE seizures, TLE, autism, and schizophrenia. Therefore, the use of BTN to antagonize neuronal NKCC1 with the goal to lower internal Cl(-) levels and promote GABAergic mediated hyperpolarization has been proposed. In this review, we summarize the data and results for pre-clinical and clinical studies that have tested off-label BTN interventions and report variable outcomes. We also compare the data underlying the developmental expression profile of NKCC1 and KCC2, highlight the limitations of BTN's brain-availability and consider its actions on non-neuronal cells.
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    Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have crit. roles in regulating cell vol., controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we detd. the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiol. functions of CCCs and interpreting disease-related mutations.
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    The secondary active cation-chloride cotransporters (CCCs) utilize the existing Na+ and/or K+ gradients to move Cl- into or out of cells. NKCC1 is an intensively studied member of the CCC family and plays fundamental roles in regulating trans-epithelial ion movement, cell vol., chloride homeostasis and neuronal excitability. Here, we report a cryo-EM structure of human NKCC1 captured in a partially loaded, inward-open state. NKCC1 assembles into a dimer, with the first ten transmembrane (TM) helixes harboring the transport core and TM11-TM12 helixes lining the dimer interface. TM1 and TM6 helixes break α-helical geometry halfway across the lipid bilayer where ion binding sites are organized around these discontinuous regions. NKCC1 may harbor multiple extracellular entryways and intracellular exits, raising the possibility that K+, Na+, and Cl- ions may traverse along their own routes for translocation. NKCC1 structure provides a blueprint for further probing structure-function relationships of NKCC1 and other CCCs.
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    The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2
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    Communications Biology (2021), 4 (1), 226CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
    NKCC and KCC transporters mediate coupled transport of Na++K++Cl- and K++Cl- across the plasma membrane, thus regulating cell Cl- concn. and cell vol. and playing crit. roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational anal. and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.
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    Zhao, Y.; Roy, K.; Vidossich, P.; Cancedda, L.; De Vivo, M.; Forbush, B.; Cao, E. Structural Basis for Inhibition of the Cation-Chloride Cotransporter NKCC1 by the Diuretic Drug Bumetanide. Nat. Commun. 2022, 13 (1), 2747,  DOI: 10.1038/s41467-022-30407-3
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    Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide
    Zhao, Yongxiang; Roy, Kasturi; Vidossich, Pietro; Cancedda, Laura; De Vivo, Marco; Forbush, Biff; Cao, Erhu
    Nature Communications (2022), 13 (1), 2747CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl- across cell membranes. NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons and NKCC2 is crit. to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with sepd. transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain assocn.
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    Krishnamurthy, Harini; Gouaux, Eric
    Nature (London, United Kingdom) (2012), 481 (7382), 469-474CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Neurotransmitter sodium symporters are integral membrane proteins that remove chem. transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homolog, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.
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    del Alamo, D.; Meiler, J.; Mchaourab, H. S. Principles of Alternating Access in LeuT-Fold Transporters: Commonalities and Divergences. J. Mol. Biol. 2022, 434 (19), 167746  DOI: 10.1016/j.jmb.2022.167746
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    del Alamo, Diego; Meiler, Jens; Mchaourab, Hassane S.
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    A review. Found in all domains of life, transporters belonging to the LeuT-fold class mediate the import and exchange of hydrophilic and charged compds. such as amino acids, metals, and sugar mols. Nearly two decades of studies on the eponymous bacterial transporter LeuT have yielded a library of high-resoln. snapshots of its conformational cycle linked by soln.-state exptl. data obtained from multiple techniques. In parallel, its topol. was obsd. in symporters and antiporters characterized by a spectrum of substrate specificities and coupled to gradients of distinct ions. Here the authors review and compare mechanistic models of transport for LeuT, its well-studied homologs, as well as functionally distant members of the fold, emphasizing the commonalities and divergences in alternating access and the corresponding energy landscapes. The authors' integrated summary illustrates how fold conservation, a hallmark of the LeuT fold, coincides with divergent choreogs. of alternating access that nevertheless capitalize on recurrent structural motifs. In addn., it highlights the knowledge gap that hinders the leveraging of the current body of research into detailed mechanisms of transport for this important class of membrane proteins.
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    Bonati, L.; Rizzi, V.; Parrinello, M. Data-Driven Collective Variables for Enhanced Sampling. J. Phys. Chem. Lett. 2020, 11 (8), 2998– 3004,  DOI: 10.1021/acs.jpclett.0c00535
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    18
    Data-Driven Collective Variables for Enhanced Sampling
    Bonati, Luigi; Rizzi, Valerio; Parrinello, Michele
    Journal of Physical Chemistry Letters (2020), 11 (8), 2998-3004CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    Designing an appropriate set of collective variables is crucial to the success of several enhanced sampling methods. Here we focus on how to obtain such variables from information limited to the metastable states. We characterize these states by a large set of descriptors and employ neural networks to compress this information in a lower-dimensional space, using Fisher's linear discriminant as an objective function to maximize the discriminative power of the network. We test this method on alanine dipeptide, using the nonlinearly separable data set composed by at. distances. We then study an intermol. aldol reaction characterized by a concerted mechanism. The resulting variables are able to promote sampling by drawing nonlinear paths in the phys. space connecting the fluctuations between metastable basins. Lastly, we interpret the behavior of the neural network by studying its relation to the phys. variables. Through the identification of its most relevant features, we are able to gain chem. insight into the process.
  19. 19
    Invernizzi, M.; Parrinello, M. Exploration vs Convergence Speed in Adaptive-Bias Enhanced Sampling. J. Chem. Theory Comput. 2022, 18 (6), 3988– 3996,  DOI: 10.1021/acs.jctc.2c00152
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    19
    Exploration vs Convergence Speed in Adaptive-Bias Enhanced Sampling
    Invernizzi, Michele; Parrinello, Michele
    Journal of Chemical Theory and Computation (2022), 18 (6), 3988-3996CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    In adaptive-bias enhanced sampling methods, a bias potential is added to the system to drive transitions between metastable states. The bias potential is a function of a few collective variables and is gradually modified according to the underlying free energy surface. We show that when the collective variables are suboptimal, there is an exploration-convergence tradeoff, and one must choose between a quickly converging bias that will lead to fewer transitions or a slower to converge bias that can explore the phase space more efficiently but might require a much longer time to produce an accurate free energy est. The recently proposed on-the-fly probability enhanced sampling (OPES) method focuses on fast convergence, but there are cases where fast exploration is preferred instead. For this reason, we introduce a new variant of the OPES method that focuses on quickly escaping metastable states at the expense of convergence speed. We illustrate the benefits of this approach in prototypical systems and show that it outperforms the popular metadynamics method.
  20. 20
    Invernizzi, M.; Parrinello, M. Rethinking Metadynamics: From Bias Potentials to Probability Distributions. J. Phys. Chem. Lett. 2020, 11 (7), 2731– 2736,  DOI: 10.1021/acs.jpclett.0c00497
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    20
    Rethinking Metadynamics: From Bias Potentials to Probability Distributions
    Invernizzi, Michele; Parrinello, Michele
    Journal of Physical Chemistry Letters (2020), 11 (7), 2731-2736CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    Metadynamics is an enhanced sampling method of great popularity, based on the on-the-fly construction of a bias potential that is a function of a selected no. of collective variables. We propose here a change in perspective that shifts the focus from the bias to the probability distribution reconstruction while retaining some of the key characteristics of metadynamics, such as flexible on-the-fly adjustments to the free energy est. The result is an enhanced sampling method that presents a drastic improvement in convergence speed, esp. when dealing with suboptimal and/or multidimensional sets of collective variables. The method is esp. robust and easy to use and in fact requires only a few simple parameters to be set, and it has a straightforward reweighting scheme to recover the statistics of the unbiased ensemble. Furthermore, it gives more control of the desired exploration of the phase space since the deposited bias is not allowed to grow indefinitely and it does not push the simulation to uninteresting high free energy regions. We demonstrate the performance of the method in a no. of representative examples.
  21. 21
    Yamashita, A.; Singh, S. K.; Kawate, T.; Jin, Y.; Gouaux, E. Crystal Structure of a Bacterial Homologue of Na+/Cl--Dependent Neurotransmitter Transporters. Nature 2005, 437 (7056), 215– 223,  DOI: 10.1038/nature03978
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    21
    Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters
    Yamashita, Atsuko; Singh, Satinder K.; Kawate, Toshimitsu; Jin, Yan; Gouaux, Eric
    Nature (London, United Kingdom) (2005), 437 (7056), 215-223CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Na+/Cl--dependent transporters terminate synaptic transmission by using electrochem. gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compds., and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homolog of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helixes, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.
  22. 22
    Forrest, L. R.; Rudnick, G. The Rocking Bundle: A Mechanism for Ion-Coupled Solute Flux by Symmetrical Transporters. Physiology 2009, 24 (6), 377– 386,  DOI: 10.1152/physiol.00030.2009
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    22
    The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters
    Forrest, Lucy R.; Rudnick, Gary
    Physiology (2009), 24 (Dec.), 377-386CODEN: PHYSCI; ISSN:1548-9213. (International Union of Physiological Sciences)
    A review. Crystal structures of the bacterial amino acid transporter LeuT have provided the basis for understanding the conformational changes assocd. with substrate translocation by a multitude of transport proteins with the same fold. Biochem. and modeling studies led to a "rocking bundle" mechanism for LeuT that was validated by subsequent transporter structures. These advances suggest how coupled solute transport might be defined by the internal symmetry of proteins contg. inverted structural repeats.
  23. 23
    Masrati, G.; Mondal, R.; Rimon, A.; Kessel, A.; Padan, E.; Lindahl, E.; Ben-Tal, N. An Angular Motion of a Conserved Four-Helix Bundle Facilitates Alternating Access Transport in the TtNapA and EcNhaA Transporters. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (50), 31850– 31860,  DOI: 10.1073/pnas.2002710117
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    23
    An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters
    Masrati, Gal; Mondal, Ramakanta; Rimon, Abraham; Kessel, Amit; Padan, Etana; Lindahl, Erik; Ben-Tal, Nir
    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (50), 31850-31860CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, resp. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using crosslinking expts.
  24. 24
    Borgogno, M.; Savardi, A.; Manigrasso, J.; Turci, A.; Portioli, C.; Ottonello, G.; Bertozzi, S. M.; Armirotti, A.; Contestabile, A.; Cancedda, L.; De Vivo, M. Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome. J. Med. Chem. 2021, 64 (14), 10203– 10229,  DOI: 10.1021/acs.jmedchem.1c00603
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    24
    Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome
    Borgogno, Marco; Savardi, Annalisa; Manigrasso, Jacopo; Turci, Alessandra; Portioli, Corinne; Ottonello, Giuliana; Bertozzi, Sine Mandrup; Armirotti, Andrea; Contestabile, Andrea; Cancedda, Laura; De Vivo, Marco
    Journal of Medicinal Chemistry (2021), 64 (14), 10203-10229CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Intracellular chloride concn. [Cl-]i is defective in several neurol. disorders. In neurons, [Cl-]i is mainly regulated by the action of the Na+-K+-Cl- importer NKCC1 and the K+-Cl- exporter KCC2. Recently, we have reported the discovery of ARN23746(I) as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chem. class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivs. In particular, we present compd. 40 (ARN24092)(II) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compds. for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.
  25. 25
    Savardi, A.; Borgogno, M.; Narducci, R.; La Sala, G.; Ortega, J. A.; Summa, M.; Armirotti, A.; Bertorelli, R.; Contestabile, A.; De Vivo, M.; Cancedda, L. Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders. Chem. 2020, 6 (8), 2073– 2096,  DOI: 10.1016/j.chempr.2020.06.017
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    25
    Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders
    Savardi, Annalisa; Borgogno, Marco; Narducci, Roberto; La Sala, Giuseppina; Ortega, Jose Antonio; Summa, Maria; Armirotti, Andrea; Bertorelli, Rosalia; Contestabile, Andrea; De Vivo, Marco; Cancedda, Laura
    Chem (2020), 6 (8), 2073-2096CODEN: CHEMVE; ISSN:2451-9294. (Cell Press)
    Aberrant expression ratio of Cl- transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clin. trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl- transporter NKCC2, creating crit. drug compliance issues and health concerns. Here, we report the discovery of a new chem. class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiol. intracellular Cl- in murine Down syndrome neuronal cultures, has excellent soly. and metabolic stability, and displays no issues with off-target activity in vitro. ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclin./manufg. studies toward the first sustainable therapeutics for the neurol. conditions characterized by impaired Cl- homeostasis.
  26. 26
    Savardi, A.; Patricelli Malizia, A.; De Vivo, M.; Cancedda, L.; Borgogno, M. Preclinical Development of the Na-K-2Cl Co-Transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders. ACS Pharmacol. Transl. Sci. 2023, 6 (1), 1– 11,  DOI: 10.1021/acsptsci.2c00197
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    26
    Preclinical Development of the Na-K-2Cl Co-transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders
    Savardi, Annalisa; Patricelli Malizia, Andrea; De Vivo, Marco; Cancedda, Laura; Borgogno, Marco
    ACS Pharmacology & Translational Science (2023), 6 (1), 1-11CODEN: APTSFN; ISSN:2575-9108. (American Chemical Society)
    Alterations in the expression of the Cl- importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl co-transporter 2 (KCC2) lead to impaired intracellular chloride concn. in neurons and imbalanced excitation/inhibition in the brain. These alterations have been obsd. in several neurol. disorders (e.g., Down syndrome and autism). Recently, we have reported the discovery of the selective NKCC1 inhibitor "compd. ARN23746" for the treatment of Down syndrome and autism in mouse models. Here, we report on an extensive preclin. characterization of ARN23746 toward its development as a clin. candidate. ARN23746 shows an overall excellent metab. profile and good brain penetration. Moreover, ARN23746 is effective in rescuing cognitive impairment in Down syndrome mice upon per os administration, in line with oral treatment of neurodevelopmental disorders. Notably, ARN23746 does not present signs of toxicity or diuresis even if administered up to 50 times the ED. These results further support ARN23746 as a solid candidate for clin. trial-enabling studies.
  27. 27
    Chew, T. A.; Zhang, J.; Feng, L. High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters. J. Mol. Biol. 2021, 433 (16), 167056  DOI: 10.1016/j.jmb.2021.167056
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    27
    High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters
    Chew, Thomas A.; Zhang, Jinru; Feng, Liang
    Journal of Molecular Biology (2021), 433 (16), 167056CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    A review. Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl- with K+ and/or Na+ in an electroneutral manner. They play important roles in myriad fundamental physiol. processes--from cell vol. regulation to transepithelial solute transport and intracellular ion homeostasis--and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacol. of these transporters, there have been several breakthroughs in the structural detn. of CCC transporters. The insights provided by these new structures for the Na+/K+/Cl- cotransporter NKCC1 and the K+/Cl- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their mol. basis and transport function. This focused discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.
  28. 28
    Lytle, C.; McManus, T. J.; Haas, M. A Model of Na-K-2Cl Cotransport Based on Ordered Ion Binding and Glide Symmetry. Am. J. Physiol.-Cell Physiol. 1998, 274 (2), C299– C309,  DOI: 10.1152/ajpcell.1998.274.2.C299
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    Delpire, E.; Gagnon, K. B. Na + -K + −2Cl – Cotransporter (NKCC) Physiological Function in Nonpolarized Cells and Transporting Epithelia. In Comprehensive Physiology; Terjung, R., Ed.; Wiley, 2018; pp. 871– 901.
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    There is no corresponding record for this reference.
  30. 30
    Shimamura, T.; Weyand, S.; Beckstein, O.; Rutherford, N. G.; Hadden, J. M.; Sharples, D.; Sansom, M. S. P.; Iwata, S.; Henderson, P. J. F.; Cameron, A. D. Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1. Science 2010, 328 (5977), 470– 473,  DOI: 10.1126/science.1186303
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    Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1
    Shimamura, Tatsuro; Weyand, Simone; Beckstein, Oliver; Rutherford, Nicholas G.; Hadden, Jonathan M.; Sharples, David; Sansom, Mark S. P.; Iwata, So; Henderson, Peter J. F.; Cameron, Alexander D.
    Science (Washington, DC, United States) (2010), 328 (5977), 470-473CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens is comprised of a five-helix inverted repeat, a conformation widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resoln., which complements previously described structures of Mhp1 in outward-facing and occluded states. From analyses of the three structures and mol. dynamics (MD) simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helixes 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
  31. 31
    Hamann, S.; Herrera-Perez, J. J.; Zeuthen, T.; Alvarez-Leefmans, F. J. Cotransport of Water by the Na + -K + −2Cl – Cotransporter NKCC1 in Mammalian Epithelial Cells: Cotransport of Water by NKCC1. J. Physiol. 2010, 588 (21), 4089– 4101,  DOI: 10.1113/jphysiol.2010.194738
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    Cotransport of water by the Na+-K+-2Cl- cotransporter NKCC1 in mammalian epithelial cells
    Hamann, Steffen; Herrera-Perez, Jose J.; Zeuthen, Thomas; Alvarez-Leefmans, Francisco J.
    Journal of Physiology (Oxford, United Kingdom) (2010), 588 (21), 4089-4101CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
    Water transport by the Na+-K+-2Cl- cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl- fluxes mediated by NKCC1 was inferred from changes in cell water vol., monitored by intracellular self-quenching of the fluorescent dye calcein. Isosmotic removal of external Cl- or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μM). When returned to the control soln. there was a rapid water influx that required the simultaneous presence of external Na+ and Cl-. The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl-] satd. in a sigmoidal fashion with a Km of 60 mM, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mM. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their vol. independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and sym. cells.
  32. 32
    Zeuthen, T.; MacAulay, N. Cotransport of Water by Na + -K + −2Cl – Cotransporters Expressed in Xenopus Oocytes: NKCC1 versus NKCC2: Water Transport in NKCC. J. Physiol. 2012, 590 (5), 1139– 1154,  DOI: 10.1113/jphysiol.2011.226316
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    Cotransport of water by Na+-K+-2Cl- cotransporters expressed in Xenopus oocytes: NKCC1 versus NKCC2
    Zeuthen, Thomas; MacAulay, Nanna
    Journal of Physiology (Oxford, United Kingdom) (2012), 590 (5), 1139-1154CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
    The NKCC1 and NKCC2 isoforms of the mammalian Na+-K+-2Cl- cotransporter were expressed in Xenopus oocytes and the relation between external ion concn. and water fluxes detd. Water fluxes were detd. from changes in the oocytes vol. and ion fluxes from 86Rb+ uptake. Isotonic increases in external K+ concn. elicited abrupt inward water fluxes in NKCC1; the K+ dependence obeyed one-site kinetics with a K0.5 of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temp. dependence and could proceed uphill against an osmotic gradient of 20 mosmol L-1. A comparison between ion and water fluxes indicates that 460 water mols. are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes. Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the notion of interaction between ions and water in NKCC1 and allows for an est. of around 600 water mols. transported per turnover of the protein. Osmotic gradients did not induce water transport in NKCC2. We conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfills its role in the kidney of transporting ions but not water. The different behavior of NKCC1 and NKCC2 is discussed on the basis of recent mol. models based on studies of structural and mol. dynamics.
  33. 33
    Sadegh, C.; Xu, H.; Sutin, J.; Fatou, B.; Gupta, S.; Pragana, A.; Taylor, M.; Kalugin, P. N.; Zawadzki, M. E.; Alturkistani, O.; Shipley, F. B.; Dani, N.; Fame, R. M.; Wurie, Z.; Talati, P.; Schleicher, R. L.; Klein, E. M.; Zhang, Y.; Holtzman, M. J.; Moore, C. I.; Lin, P.-Y.; Patel, A. B.; Warf, B. C.; Kimberly, W. T.; Steen, H.; Andermann, M. L.; Lehtinen, M. K. Choroid Plexus-Targeted NKCC1 Overexpression to Treat Post-Hemorrhagic Hydrocephalus. Neuron 2023, 111 (10), 1591– 1608,  DOI: 10.1016/j.neuron.2023.02.020
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    33
    Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus
    Sadegh, Cameron; Xu, Huixin; Sutin, Jason; Fatou, Benoit; Gupta, Suhasini; Pragana, Aja; Taylor, Milo; Kalugin, Peter N.; Zawadzki, Miriam E.; Alturkistani, Osama; Shipley, Frederick B.; Dani, Neil; Fame, Ryann M.; Wurie, Zainab; Talati, Pratik; Schleicher, Riana L.; Klein, Eric M.; Zhang, Yong; Holtzman, Michael J.; Moore, Christopher I.; Lin, Pei-Yi; Patel, Aman B.; Warf, Benjamin C.; Kimberly, W. Taylor; Steen, Hanno; Andermann, Mark L.; Lehtinen, Maria K.
    Neuron (2023), 111 (10), 1591-1608.e4CODEN: NERNET; ISSN:0896-6273. (Cell Press)
    Post-hemorrhagic hydrocephalus (PHH) refers to a life-threatening accumulation of cerebrospinal fluid (CSF) that occurs following intraventricular hemorrhage (IVH). An incomplete understanding of this variably progressive condition has hampered the development of new therapies beyond serial neurosurgical interventions. Here, we show a key role for the bidirectional Na-K-Cl cotransporter, NKCC1, in the choroid plexus (ChP) to mitigate PHH. Mimicking IVH with intraventricular blood led to increased CSF [K+] and triggered cytosolic calcium activity in ChP epithelial cells, which was followed by NKCC1 activation. ChP-targeted adeno-assocd. viral (AAV)-NKCC1 prevented blood-induced ventriculomegaly and led to persistently increased CSF clearance capacity. These data demonstrate that intraventricular blood triggered a trans-choroidal, NKCC1-dependent CSF clearance mechanism. Inactive, phosphodeficient AAV-NKCC1-NT51 failed to mitigate ventriculomegaly. Excessive CSF [K+] fluctuations correlated with permanent shunting outcome in humans following hemorrhagic stroke, suggesting targeted gene therapy as a potential treatment to mitigate intracranial fluid accumulation following hemorrhage.
  34. 34
    Steffensen, A. B.; Oernbo, E. K.; Stoica, A.; Gerkau, N. J.; Barbuskaite, D.; Tritsaris, K.; Rose, C. R.; MacAulay, N. Cotransporter-Mediated Water Transport Underlying Cerebrospinal Fluid Formation. Nat. Commun. 2018, 9 (1), 2167,  DOI: 10.1038/s41467-018-04677-9
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    Cotransporter-mediated water transport underlying cerebrospinal fluid formation
    Steffensen Annette B; Oernbo Eva K; Stoica Anca; MacAulay Nanna; Gerkau Niklas J; Rose Christine R; Barbuskaite Dagne; Tritsaris Katerina
    Nature communications (2018), 9 (1), 2167 ISSN:.
    Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na(+)/K(+)/2Cl(-) cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.
  35. 35
    Li, J.; Shaikh, S. A.; Enkavi, G.; Wen, P.-C.; Huang, Z.; Tajkhorshid, E. Transient Formation of Water-Conducting States in Membrane Transporters. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (19), 7696– 7701,  DOI: 10.1073/pnas.1218986110
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    Transient formation of water-conducting states in membrane transporters
    Li, Jing; Shaikh, Saher A.; Enkavi, Giray; Wen, Po-Chao; Huang, Zhijian; Tajkhorshid, Emad
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (19), 7696-7701, S7696/1-S7696/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane - a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mech. coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equil. mol. dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying mol. mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the obsd. water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.
  36. 36
    Okazaki, K.; Wöhlert, D.; Warnau, J.; Jung, H.; Yildiz, Ö.; Kühlbrandt, W.; Hummer, G. Mechanism of the Electroneutral Sodium/Proton Antiporter PaNhaP from Transition-Path Shooting. Nat. Commun. 2019, 10 (1), 1742,  DOI: 10.1038/s41467-019-09739-0
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    Mechanism of the electroneutral sodium/proton antiporter PaNhaP from transition-path shooting
    Okazaki Kei-Ichi; Okazaki Kei-Ichi; Warnau Judith; Jung Hendrik; Hummer Gerhard; Wohlert David; Yildiz Ozkan; Kuhlbrandt Werner; Hummer Gerhard
    Nature communications (2019), 10 (1), 1742 ISSN:.
    Na(+)/H(+) antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na(+)/H(+) antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na(+)/H(+) exchanger NHE1, which is an important drug target. Here we resolve the Na(+) and H(+) transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.
  37. 37
    Monette, M. Y.; Somasekharan, S.; Forbush, B. Molecular Motions Involved in Na-K-Cl Cotransporter-Mediated Ion Transport and Transporter Activation Revealed by Internal Cross-Linking between Transmembrane Domains 10 and 11/12. J. Biol. Chem. 2014, 289 (11), 7569– 7579,  DOI: 10.1074/jbc.M113.542258
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    Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Cross-linking between Transmembrane Domains 10 and 11/12
    Monette, Michelle Y.; Somasekharan, Suma; Forbush, Biff
    Journal of Biological Chemistry (2014), 289 (11), 7569-7579CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    We examd. the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homol. models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function; indeed, evidence for cross-link formation with low micromolar concns. of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 addnl. tested pairs. Inhibition of transport was obsd. with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu2+ inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met382 of TM3 are involved in a Cu2+-binding site. Surprisingly, crosslinking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) crosslinking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by crosslinking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.
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    McNeill, A.; Iovino, E.; Mansard, L.; Vache, C.; Baux, D.; Bedoukian, E.; Cox, H.; Dean, J.; Goudie, D.; Kumar, A.; Newbury-Ecob, R.; Fallerini, C.; Renieri, A.; Lopergolo, D.; Mari, F.; Blanchet, C.; Willems, M.; Roux, A.-F.; Pippucci, T.; Delpire, E. SLC12A2 Variants Cause a Neurodevelopmental Disorder or Cochleovestibular Defect. Brain 2020, 143 (8), 2380– 2387,  DOI: 10.1093/brain/awaa176
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    SLC12A2 variants cause a neurodevelopmental disorder or cochleovestibular defect
    McNeill Alisdair; McNeill Alisdair; McNeill Alisdair; Iovino Emanuela; Mansard Luke; Vache Christel; Baux David; Roux Anne-Francoise; Bedoukian Emma; Cox Helen; Dean John; Goudie David; Kumar Ajith; Newbury-Ecob Ruth; Fallerini Chiara; Renieri Alessandra; Lopergolo Diego; Mari Francesca; Fallerini Chiara; Renieri Alessandra; Lopergolo Diego; Mari Francesca; Blanchet Catherine; Willems Marjolaine; Pippucci Tommaso; Delpire Eric
    Brain : a journal of neurology (2020), 143 (8), 2380-2387 ISSN:.
    The SLC12 gene family consists of SLC12A1-SLC12A9, encoding electroneutral cation-coupled chloride co-transporters. SCL12A2 has been shown to play a role in corticogenesis and therefore represents a strong candidate neurodevelopmental disorder gene. Through trio exome sequencing we identified de novo mutations in SLC12A2 in six children with neurodevelopmental disorders. All had developmental delay or intellectual disability ranging from mild to severe. Two had sensorineural deafness. We also identified SLC12A2 variants in three individuals with non-syndromic bilateral sensorineural hearing loss and vestibular areflexia. The SLC12A2 de novo mutation rate was demonstrated to be significantly elevated in the deciphering developmental disorders cohort. All tested variants were shown to reduce co-transporter function in Xenopus laevis oocytes. Analysis of SLC12A2 expression in foetal brain at 16-18 weeks post-conception revealed high expression in radial glial cells, compatible with a role in neurogenesis. Gene co-expression analysis in cells robustly expressing SLC12A2 at 16-18 weeks post-conception identified a transcriptomic programme associated with active neurogenesis. We identify SLC12A2 de novo mutations as the cause of a novel neurodevelopmental disorder and bilateral non-syndromic sensorineural hearing loss and provide further data supporting a role for this gene in human neurodevelopment.
  39. 39
    Janoš, P.; Magistrato, A. All-Atom Simulations Uncover the Molecular Terms of the NKCC1 Transport Mechanism. J. Chem. Inf. Model. 2021, 61 (7), 3649– 3658,  DOI: 10.1021/acs.jcim.1c00551
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    All-Atom Simulations Uncover the Molecular Terms of the NKCC1 Transport Mechanism
    Janos, Pavel; Magistrato, Alessandra
    Journal of Chemical Information and Modeling (2021), 61 (7), 3649-3658CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    The secondary-active Na-K-Cl cotransporter 1 (NKCC1), member of the cation-chloride cotransporter (CCC) family, ensures the electroneutral movement of Cl-, Na+, and K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell vol., handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, and cancer). Building on the newly resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ion transport, assessing the order and the mol. basis of its interdependent ion translocation. The authors' outcomes strikingly advance the understanding of the physiol. mechanism of CCCs and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both neg. and pos. charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis.
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    Portioli, C.; Ruiz Munevar, M. J.; De Vivo, M.; Cancedda, L. Cation-Coupled Chloride Cotransporters: Chemical Insights and Disease Implications. Trends Chem. 2021, 3 (10), 832– 849,  DOI: 10.1016/j.trechm.2021.05.004
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    Cation-coupled chloride cotransporters: chemical insights and disease implications
    Portioli, Corinne; Ruiz Munevar, Manuel Jose; De Vivo, Marco; Cancedda, Laura
    Trends in Chemistry (2021), 3 (10), 832-849CODEN: TCRHBQ; ISSN:2589-5974. (Cell Press)
    A review. Cation-coupled chloride cotransporters (CCCs) modulate the transport of sodium and/or potassium cations coupled with chloride anions across the cell membrane. CCCs thus help regulate intracellular ionic concn. and consequent cell vol. homeostasis. This has been largely exploited in the past to develop diuretic drugs that act on CCCs expressed in the kidney. However, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. Here, we examine the structure-function relationship of CCCs. By linking recent high-resoln. cryogenic electron microscopy (cryo-EM) data with older biochem./functional studies on CCCs, we discuss the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies.
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    Zhao, Y.; Shen, J.; Wang, Q.; Ruiz Munevar, M. J.; Vidossich, P.; De Vivo, M.; Zhou, M.; Cao, E. Structure of the Human Cation–Chloride Cotransport KCC1 in an Outward-Open State. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (27), e2109083119  DOI: 10.1073/pnas.2109083119
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    Structure of the human cation-chloride cotransport KCC1 in an outward-open state
    Zhao, Yongxiang; Shen, Jiemin; Wang, Qinzhe; Munevar, Manuel Jose Ruiz; Vidossich, Pietro; De Vivo, Marco; Zhou, Ming; Cao, Erhu
    Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (27), e2109083119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell vol. homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concn., and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helixes. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small mol. compds.
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    Saitsu, H.; Watanabe, M.; Akita, T.; Ohba, C.; Sugai, K.; Ong, W. P.; Shiraishi, H.; Yuasa, S.; Matsumoto, H.; Beng, K. T.; Saitoh, S.; Miyatake, S.; Nakashima, M.; Miyake, N.; Kato, M.; Fukuda, A.; Matsumoto, N. Impaired Neuronal KCC2 Function by Biallelic SLC12A5Mutations in Migrating Focal Seizures and Severe Developmental Delay. Sci. Rep. 2016, 6 (1), 30072,  DOI: 10.1038/srep30072
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    Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay
    Saitsu, Hirotomo; Watanabe, Miho; Akita, Tenpei; Ohba, Chihiro; Sugai, Kenji; Ong, Winnie Peitee; Shiraishi, Hideaki; Yuasa, Shota; Matsumoto, Hiroshi; Beng, Khoo Teik; Saitoh, Shinji; Miyatake, Satoko; Nakashima, Mitsuko; Miyake, Noriko; Kato, Mitsuhiro; Fukuda, Atsuo; Matsumoto, Naomichi
    Scientific Reports (2016), 6 (), 30072CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compd. heterozygous SLC12A5 (encoding the neuronal K+-Cl- co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compd. heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp anal. demonstrated strongly suppressed Cl- extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl- level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl- extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS.
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    Uyanik, G.; Elcioglu, N.; Penzien, J.; Gross, C.; Yilmaz, Y.; Olmez, A.; Demir, E.; Wahl, D.; Scheglmann, K.; Winner, B.; Bogdahn, U.; Topaloglu, H.; Hehr, U.; Winkler, J. Novel Truncating and Missense Mutations of the KCC3 Gene Associated with Andermann Syndrome. Neurology 2006, 67 (7), 1044,  DOI: 10.1212/01.wnl.0000250608.09509.ed
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    Olsson, M. H. M.; So̷ndergaard, C. R.; Rostkowski, M.; Jensen, J. H. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p Ka Predictions. J. Chem. Theory Comput. 2011, 7 (2), 525– 537,  DOI: 10.1021/ct100578z
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    PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions
    Olsson, Mats H. M.; Sondergaard, Chresten R.; Rostkowski, Michal; Jensen, Jan H.
    Journal of Chemical Theory and Computation (2011), 7 (2), 525-537CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    The authors have revised the rules and parameters for one of the most commonly used empirical pKa predictors, PROPKA, based on better phys. description of the desolvation and dielec. response for the protein. The authors have introduced a new and consistent approach to interpolate the description between the previously distinct classifications into internal and surface residues, which otherwise is found to give rise to an erratic and discontinuous behavior. Since the goal of this study is to lay out the framework and validate the concept, it focuses on Asp and Glu residues where the protein pKa values and structures are assumed to be more reliable. The new and improved implementation is evaluated and discussed; it is found to agree better with expt. than the previous implementation (in parentheses): rmsd = 0.79 (0.91) for Asp and Glu, 0.75 (0.97) for Tyr, 0.65 (0.72) for Lys, and 1.00 (1.37) for His residues. The most significant advance, however, is in reducing the no. of outliers and removing unreasonable sensitivity to small structural changes that arise from classifying residues as either internal or surface.
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    Schott-Verdugo, S.; Gohlke, H. PACKMOL-Memgen: A Simple-To-Use, Generalized Workflow for Membrane-Protein–Lipid-Bilayer System Building. J. Chem. Inf. Model. 2019, 59 (6), 2522– 2528,  DOI: 10.1021/acs.jcim.9b00269
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    PACKMOL-Memgen: A Simple-To-Use, Generalized Workflow for Membrane-Protein-Lipid-Bilayer System Building
    Schott-Verdugo, Stephan; Gohlke, Holger
    Journal of Chemical Information and Modeling (2019), 59 (6), 2522-2528CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    We present PACKMOL-Memgen, a simple-to-use, generalized workflow for automated building of membrane-protein-lipid-bilayer systems based on open-source tools including Packmol, memembed, pdbremix, and AmberTools. Compared with web-interface-based related tools, PACKMOL-Memgen allows setup of multiple configurations of a system in a user-friendly and efficient manner within minutes. The generated systems are well-packed and thus well-suited as starting configurations in MD simulations under periodic boundary conditions, requiring only moderate equilibration times. PACKMOL-Memgen is distributed with AmberTools and runs on most computing platforms, and its output can also be used for CHARMM or adapted to other mol.-simulation packages.
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    Le Grand, S.; Götz, A. W.; Walker, R. C. SPFP: Speed without Compromise─A Mixed Precision Model for GPU Accelerated Molecular Dynamics Simulations. Comput. Phys. Commun. 2013, 184 (2), 374– 380,  DOI: 10.1016/j.cpc.2012.09.022
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    SPFP: Speed without compromise-A mixed precision model for GPU accelerated molecular dynamics simulations
    Le Grand, Scott; Gotz, Andreas W.; Walker, Ross C.
    Computer Physics Communications (2013), 184 (2), 374-380CODEN: CPHCBZ; ISSN:0010-4655. (Elsevier B.V.)
    A new precision model is proposed for the acceleration of all-atom classical mol. dynamics (MD) simulations on graphics processing units (GPUs). This precision model replaces double precision arithmetic with fixed point integer arithmetic for the accumulation of force components as compared to a previously introduced model that uses mixed single/double precision arithmetic. This significantly boosts performance on modern GPU hardware without sacrificing numerical accuracy. We present an implementation for NVIDIA GPUs of both generalized Born implicit solvent simulations as well as explicit solvent simulations using the particle mesh Ewald (PME) algorithm for long-range electrostatics using this precision model. Tests demonstrate both the performance of this implementation as well as its numerical stability for const. energy and const. temp. biomol. MD as compared to a double precision CPU implementation and double and mixed single/double precision GPU implementations.
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    Salomon-Ferrer, R.; Case, D. A.; Walker, R. C. An Overview of the Amber Biomolecular Simulation Package: Amber Biomolecular Simulation Package. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2013, 3 (2), 198– 210,  DOI: 10.1002/wcms.1121
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    An overview of the amber biomolecular simulation package
    Salomon-Ferrer, Romelia; Case, David A.; Walker, Ross C.
    Wiley Interdisciplinary Reviews: Computational Molecular Science (2013), 3 (2), 198-210CODEN: WIRCAH; ISSN:1759-0884. (Wiley-Blackwell)
    A review. Mol. dynamics (MD) allows the study of biol. and chem. systems at the atomistic level on timescales from femtoseconds to milliseconds. It complements expt. while also offering a way to follow processes difficult to discern with exptl. techniques. Numerous software packages exist for conducting MD simulations of which one of the widest used is termed Amber. Here, we outline the most recent developments, since version 9 was released in Apr. 2006, of the Amber and AmberTools MD software packages, referred to here as simply the Amber package. The latest release represents six years of continued development, since version 9, by multiple research groups and the culmination of over 33 years of work beginning with the first version in 1979. The latest release of the Amber package, version 12 released in Apr. 2012, includes a substantial no. of important developments in both the scientific and computer science arenas. We present here a condensed vision of what Amber currently supports and where things are likely to head over the coming years. Figure 1 shows the performance in ns/day of the Amber package version 12 on a single-core AMD FX-8120 8-Core 3.6GHz CPU, the Cray XT5 system, and a single GPU GTX680.
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    Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
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    ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
    Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
    Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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    Dickson, C. J.; Madej, B. D.; Skjevik, Å. A.; Betz, R. M.; Teigen, K.; Gould, I. R.; Walker, R. C. Lipid14: The Amber Lipid Force Field. J. Chem. Theory Comput. 2014, 10 (2), 865– 879,  DOI: 10.1021/ct4010307
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    Lipid14: The Amber Lipid Force Field
    Dickson, Callum J.; Madej, Benjamin D.; Skjevik, Age A.; Betz, Robin M.; Teigen, Knut; Gould, Ian R.; Walker, Ross C.
    Journal of Chemical Theory and Computation (2014), 10 (2), 865-879CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    The AMBER lipid force field has been updated to create Lipid14, allowing tensionless simulation of a no. of lipid types with the AMBER MD package. The modular nature of this force field allows numerous combinations of head and tail groups to create different lipid types, enabling the easy insertion of new lipid species. The Lennard-Jones and torsion parameters of both the head and tail groups have been revised and updated partial charges calcd. The force field has been validated by simulating bilayers of six different lipid types for a total of 0.5 μs each without applying a surface tension; with favorable comparison to expt. for properties such as area per lipid, vol. per lipid, bilayer thickness, NMR order parameters, scattering data, and lipid lateral diffusion. As the derivation of this force field is consistent with the AMBER development philosophy, Lipid14 is compatible with the AMBER protein, nucleic acid, carbohydrate, and small mol. force fields.
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    Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79 (2), 926– 935,  DOI: 10.1063/1.445869
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    Comparison of simple potential functions for simulating liquid water
    Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.; Impey, Roger W.; Klein, Michael L.
    Journal of Chemical Physics (1983), 79 (2), 926-35CODEN: JCPSA6; ISSN:0021-9606.
    Classical Monte Carlo simulations were carried out for liq. H2O in the NPT ensemble at 25° and 1 atm using 6 of the simpler intermol. potential functions for the dimer. Comparisons were made with exptl. thermodn. and structural data including the neutron diffraction results of Thiessen and Narten (1982). The computed densities and potential energies agree with expt. except for the original Bernal-Fowler model, which yields an 18% overest. of the d. and poor structural results. The discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations. Comparisons were made for the self-diffusion coeffs. obtained from mol. dynamics simulations.
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    Joung, I. S.; Cheatham, T. E. Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations. J. Phys. Chem. B 2008, 112 (30), 9020– 9041,  DOI: 10.1021/jp8001614
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    Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations
    Joung, In Suk; Cheatham, Thomas E.
    Journal of Physical Chemistry B (2008), 112 (30), 9020-9041CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    Alkali (Li+, Na+, K+, Rb+, and Cs+) and halide (F-, Cl-, Br-, and I-) ions play an important role in many biol. phenomena, roles that range from stabilization of biomol. structure, to influence on biomol. dynamics, to key physiol. influence on homeostasis and signaling. To properly model ionic interaction and stability in atomistic simulations of biomol. structure, dynamics, folding, catalysis, and function, an accurate model or representation of the monovalent ions is critically necessary. A good model needs to simultaneously reproduce many properties of ions, including their structure, dynamics, solvation, and moreover both the interactions of these ions with each other in the crystal and in soln. and the interactions of ions with other mols. At present, the best force fields for biomols. employ a simple additive, nonpolarizable, and pairwise potential for at. interaction. In this work, the authors describe their efforts to build better models of the monovalent ions within the pairwise Coulombic and 6-12 Lennard-Jones framework, where the models are tuned to balance crystal and soln. properties in Ewald simulations with specific choices of well-known water models. Although it has been clearly demonstrated that truly accurate treatments of ions will require inclusion of nonadditivity and polarizability (particularly with the anions) and ultimately even a quantum mech. treatment, the authors' goal was to simply push the limits of the additive treatments to see if a balanced model could be created. The applied methodol. is general and can be extended to other ions and to polarizable force-field models. The authors' starting point centered on observations from long simulations of biomols. in salt soln. with the AMBER force fields where salt crystals formed well below their soly. limit. The likely cause of the artifact in the AMBER parameters relates to the naive mixing of the Smith and Dang chloride parameters with AMBER-adapted Aqvist cation parameters. To provide a more appropriate balance, the authors reoptimized the parameters of the Lennard-Jones potential for the ions and specific choices of water models. To validate and optimize the parameters, the authors calcd. hydration free energies of the solvated ions and also lattice energies (LE) and lattice consts. (LC) of alkali halide salt crystals. This is the first effort that systematically scans across the Lennard-Jones space (well depth and radius) while balancing ion properties like LE and LC across all pair combinations of the alkali ions and halide ions. The optimization across the entire monovalent series avoids systematic deviations. The ion parameters developed, optimized, and characterized were targeted for use with some of the most commonly used rigid and nonpolarizable water models, specifically TIP3P, TIP4PEW, and SPC/E. In addn. to well reproducing the soln. and crystal properties, the new ion parameters well reproduce binding energies of the ions to water and the radii of the first hydration shells.
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    Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An N ·log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98 (12), 10089– 10092,  DOI: 10.1063/1.464397
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    Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems
    Darden, Tom; York, Darrin; Pedersen, Lee
    Journal of Chemical Physics (1993), 98 (12), 10089-92CODEN: JCPSA6; ISSN:0021-9606.
    An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolution using fast Fourier transforms. Timings and accuracies are presented for three large cryst. ionic systems.
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    Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23 (3), 327– 341,  DOI: 10.1016/0021-9991(77)90098-5
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    Ryckaert, Jean Paul; Ciccotti, Giovanni; Berendsen, Herman J. C.
    Journal of Computational Physics (1977), 23 (3), 327-41CODEN: JCTPAH; ISSN:0021-9991.
    A numerical algorithm integrating the 3N Cartesian equation of motion of a system of N points subject to holonomic constraints is applied to mol. dynamics simulation of a liq. of 64 butane mols.
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    Ansari, N.; Rizzi, V.; Parrinello, M. Water Regulates the Residence Time of Benzamidine in Trypsin. Nat. Commun. 2022, 13 (1), 5438,  DOI: 10.1038/s41467-022-33104-3
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    Water regulates the residence time of Benzamidine in Trypsin
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  1. Ayan Majumder, John E. Straub. Machine Learning Derived Collective Variables for the Study of Protein Homodimerization in Membrane. Journal of Chemical Theory and Computation 2024, 20 (13) , 5774-5783. https://doi.org/10.1021/acs.jctc.4c00454
  2. Yongxiang Zhao, Pietro Vidossich, Biff Forbush, Junfeng Ma, Jesse Rinehart, Marco De Vivo, Erhu Cao. Structural basis for human NKCC1 inhibition by loop diuretic drugs. The EMBO Journal 2025, 44 (5) , 1540-1562. https://doi.org/10.1038/s44318-025-00368-6
  3. Sam W. Henderson, Saeed Nourmohammadi, Maria Hrmova. Protein Structural Modeling and Transport Thermodynamics Reveal That Plant Cation–Chloride Cotransporters Mediate Potassium–Chloride Symport. International Journal of Molecular Sciences 2024, 25 (23) , 12955. https://doi.org/10.3390/ijms252312955
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  • Abstract

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    Figure 1

    Figure 1. NKCC1’s structure embedded in the membrane. (A) 3D representation of full-length human NKCC1 (PDB 7MXO), with each monomer represented in dark and light gray. Each monomer is constructed by three main components: (i) conserved TM domain, composed of 12 helices, which contains all four ion binding sites, shown in green, blue and orange for Cl–, Na+, and K+ ions, respectively; (ii) disordered amino-terminal; and (iii) large carboxy-terminal domains. (13) All structures of NKCC1, and related CCC’s of the same family, (40) revealed that the TM helices are organized in two inverted repeats of five-helix bundles─also known as LeuT-fold. (2,16,17,21) (B) Schematic representation of NKCC1, showing its homodimeric structure with one monomer represented to highlight the transmembrane domains (TMs, dark gray); the other monomer represented to highlight the channel arrangement across the cell membrane (light brown); the two five-helix bundle inverted repeats (TM 1 to TM 5 and TM 6 to TM 10) and the dimeric interface (TM 11 and TM 12). The amino and carboxy-terminal domains are also highlighted.

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    Figure 2

    Figure 2. Essential conformational transition for alternating accessibility of ion binding sites allows NKCC1 ion transport. (A) Schematic representation of outward open (left) and inward open (right) NKCC1 conformation. Ionic binding sites are exposed to the extra and intracellular side of the membrane, respectively. (B) In gray, atomic surface representation of outward open (left) and inward open (right) NKCC1 conformation. Ionic binding sites for Na+, K+, and Cl– are shown as colored surfaces (blue, orange, and green, respectively), and ions are represented as colored spheres. Outer (left) and inner (right) vestibules, open to the extra and intracellular sides of the membrane, are highlighted with discontinuous black lines.

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    Figure 3

    Figure 3. Rocking-bundle angular motion of specific NKCC1 TMs facilitates alternate accessibility of ion binding sites. (A) Schematic representation of NKCC1’s angular motion, defined as the change of the angle (α) between TM 4 and TM 9 (in green) and TM 2 and TM 7 (in red), during the conformational transition between the outward open state (bright green) and inward open state (dim green), calculated from the centers of mass of the backbone atoms from the extracellular and intracellular tip of TM 4 and TM 9 and the intracellular tip of TM 2 and TM 7 (in red). (B) Quantification of TM 4 and TM 9 angular motion represented by the angle α through 16 conformational transitions from OPES Explore simulations. The light brown horizontal bars represent the outward open and inward open average angle α ± 1SD calculated from 1 μs of equilibrium molecular dynamics (MD). Circles represent the starting point of each transition, whereas the triangles represent the end point of the same transition and its direction. Circles and triangles are colored depending on the NKCC1 conformation they represent (bright green for outward open and dim green for inward open).

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    Figure 4

    Figure 4. Stabilization of the hydrophobic interface between TM 4 and TM 9 allows for their cooperative action. Representation of human NKCC1 embedded in the cell membrane, with TM 4 and TM 9 highlighted in bright yellow. Inset on the right: Higher magnification of the hydrophobic interface between TM 4 and TM 9 (highlighted by the oval), which allows for their cooperative angular motion. Relevant residues are shown as sticks with their atomic surfaces pictured in red (oxygen), blue (nitrogen), gray (carbon), and white (hydrogen).

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    Figure 5

    Figure 5. NKCC1 TM 10's corking motion modulates ion/water access to the outer vestibule. (A) Schematic representation of NKCC1 TM 10's corking motion, defined as the change of TM 10's (in green) intrahelical angle (θ), during the conformational transition between the outward open state (bright green) and inward open state (dim green), calculated from the centers of mass of the backbone atoms from TM 10’s intra and extracellular tips, and the backbone atoms where TM 10 bends. (B) Quantification of TM 10's corking motion represented by the angle θ through 16 conformational transitions from OPES Explore simulations. The light brown horizontal bars represent the outward open and inward open average angle θ ± 1SD calculated from 1 μs of equilibrium molecular dynamics (MD). Circles represent the starting point of each transition, whereas the triangles represent the end point of the same transition and its direction. Circles and triangles are colored depending on the NKCC1 conformation they represent (bright green for outward open and dim green for inward open).

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    Figure 6

    Figure 6. Interface between TM 10 and TM 6 highlights crucial interactions that determine accessibility of extracellular binding sites. Representation of human NKCC1 embedded in the cell membrane, with TM 10 and TM 6 highlighted in bright yellow. Inset on the top right: In the IO state, as shown by the light green schematic representation, interacting residues at the TM 10 and TM 6 interface are shown as sticks with their atomic surfaces (pictured in red─oxygen, blue─nitrogen, gray─carbon, and white─hydrogen), blocking solvent access to the outer vestibule. Inset on the bottom right: In the OO state, as shown by the light orange schematic representation, previously interacting residues at the TM 10 and TM 6 interface are now shown to be too far apart to form bonds.

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    Figure 7

    Figure 7. Mutagenesis targeting residues from TM 10 and TM 6 highlight their functional relevance. (A) Representation of human NKCC1 embedded in the cell membrane, with TM 10 and TM 6 highlighted in bright yellow. Inset on the right: The interface between TM 10 and TM 6 where homologous mutated residues are shown as sticks and are highlighted in green surface. Namely, these residues are mouse A490W (human Ala 497), mouse L664A (human Leu 671), mouse N665A (human Asn 672), and mouse A668W (human Ala 675). Gray surface represents the position of residues that mainly form/break interactions throughout TM 10's corking motion. (B) Example traces obtained in the Cl– influx assay on HEK293 cells transfected with the WT NKCC1 transporter or NKCC1 mutated at different residues. The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC1-mediated Cl– influx. (C) Quantification of the mouse NKCC1 inhibitory activity using the Cl– influx fluorescence assay in HEK293 cells. A fluorescence signal decrease, corresponding to a decrease in NKCC1 transporter activity, was observed for all the cells transfected with NKCC1 mutants. Data are normalized and the average of the last 10 s of kinetics is plotted (ΔF/F0). Data are presented as a percentage of the WT. Data represent mean ± SEM from 3 to 4 independent experiments (Kruskal–Wallis one way ANOVA, H = 216, DF = 6, followed by Dunn’s post hoc test on multiple comparisons, *** P = 0.0002, **** P < 0.0001).

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    Figure 8

    Figure 8. Free energy surface identifies relevant NKCC1 conformations for the inward open ↔ outward open transition. (A) Representation of the Free Energy Surface of the conformational transition between human NKCC1 IO and OO states computed by OPES Explore over the DeepLDA collective variable and the outer vestibule water coordination collective variable. Energetical basins are highlighted with a schematic representation of the conformation they identify. These are, namely: the IO state, the occluded state (labeled Occ), the OOd state (outward open “dry” – lower outer vestibule hydration) and OOw (outward open “wet” – higher outer vestibule hydration. (B) Higher magnification of the hexagon in A representing the main gating interactions that occlude the outer vestibule and block solvent access to the ionic binding sites. Relevant residues are shown as sticks with their atomic surfaces pictured in red (oxygen), blue (nitrogen), gray (carbon) and white (hydrogen). (C) Higher magnification of the hexagon in A representing of the main gating interactions that occlude the inner vestibule and block solvent access to the ionic binding sites. Relevant residues are shown as sticks with their atomic surfaces pictured in red (oxygen), blue (nitrogen), yellow (sulfur), gray (carbon) and white (hydrogen).

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    Figure 9

    Figure 9. Pore profile confirms distinct binding-site accessibility of NKCC1 states along the inward open ↔ outward open transition. (A–C) Pore profile of the IO state (A), the occluded state (B), and OO state (C), schematically represented at the bottom. These profiles were obtained by calculating the radius of the largest sphere along the Z-axis of the ion translocation cavity, and then plotting the pore profile of several snapshots from their corresponding equilibrium MD simulations, computed by the software HOLE. Pore profiles were mirrored around radius 0 for visual clarity. The blue lines represent the outer vestibule, and the orange lines represent the inner vestibule of NKCC1.

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    Figure 10

    Figure 10. NKCC1’s outward open conformations are permeable to water. (A) Representation of NKCC1 (gray cartoon) embedded in the membrane (black horizontal bars) in a water-permeable state. Water molecules are shown as red and white lines with a red atomic surface representation. A chain of water molecules whose oxygen atoms are within 4.0 Å of each other connecting the extracellular and intracellular solvent is present. (B, C) Schematic representation of both states that present permeability to water (outward open without ions bound, B, or fully loaded, C), and their respective plot, which tracks the appearance of permeable states during each state’s equilibrium MD simulation. Vertical red bars represent snapshots from the respective simulation where a chain of water molecules whose oxygen atoms are within 4.0 Å of each other connecting the extracellular and intracellular solvent through NKCC1 is observed.

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    Figure 11

    Figure 11. NKCC1 passively transports water. Histogram of all transport events detected in the state equilibrium MD simulation of NKCC1 outward open conformation with no ions bound, organized by the length of each transport event. Bars show the frequency of efflux/influx (orange/green) events per transport event duration. Vertical continuous lines show the mean, and discontinuous lines show the standard deviation (efflux, orange; influx, green). Some longer transport events were excluded from the histogram for clarity.

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      Kharod Shivani C; Kang Seok Kyu; Kadam Shilpa D; Kadam Shilpa D
      Frontiers in neuroscience (2019), 13 (), 310 ISSN:1662-4548.
      Bumetanide (BTN or BUM) is a FDA-approved potent loop diuretic (LD) that acts by antagonizing sodium-potassium-chloride (Na-K-Cl) cotransporters, NKCC1 (SLc12a2) and NKCC2. While NKCC1 is expressed both in the CNS and in systemic organs, NKCC2 is kidney-specific. The off-label use of BTN to modulate neuronal transmembrane Cl(-) gradients by blocking NKCC1 in the CNS has now been tested as an anti-seizure agent and as an intervention for neurological disorders in pre-clinical studies with varying results. BTN safety and efficacy for its off-label use has also been tested in several clinical trials for neonates, children, adolescents, and adults. It failed to meet efficacy criteria for hypoxic-ischemic encephalopathy (HIE) neonatal seizures. In contrast, positive outcomes in temporal lobe epilepsy (TLE), autism, and schizophrenia trials have been attributed to BTN in studies evaluating its off-label use. NKCC1 is an electroneutral neuronal Cl(-) importer and the dominance of NKCC1 function has been proposed as the common pathology for HIE seizures, TLE, autism, and schizophrenia. Therefore, the use of BTN to antagonize neuronal NKCC1 with the goal to lower internal Cl(-) levels and promote GABAergic mediated hyperpolarization has been proposed. In this review, we summarize the data and results for pre-clinical and clinical studies that have tested off-label BTN interventions and report variable outcomes. We also compare the data underlying the developmental expression profile of NKCC1 and KCC2, highlight the limitations of BTN's brain-availability and consider its actions on non-neuronal cells.
    10. 10
      Wang, J.; Liu, R.; Hasan, M. N.; Fischer, S.; Chen, Y.; Como, M.; Fiesler, V. M.; Bhuiyan, M. I. H.; Dong, S.; Li, E.; Kahle, K. T.; Zhang, J.; Deng, X.; Subramanya, A. R.; Begum, G.; Yin, Y.; Sun, D. Role of SPAK–NKCC1 Signaling Cascade in the Choroid Plexus Blood–CSF Barrier Damage after Stroke. J. Neuroinflammation 2022, 19 (1), 91,  DOI: 10.1186/s12974-022-02456-4
      10
      Role of SPAK-NKCC1 signaling cascade in the choroid plexus blood-CSF barrier damage after stroke
      Wang, Jun; Liu, Ruijia; Hasan, Md Nabiul; Fischer, Sydney; Chen, Yang; Como, Matt; Fiesler, Victoria M.; Bhuiyan, Mohammad Iqbal H.; Dong, Shuying; Li, Eric; Kahle, Kristopher T.; Zhang, Jinwei; Deng, Xianming; Subramanya, Arohan R.; Begum, Gulnaz; Yin, Yan; Sun, Dandan
      Journal of Neuroinflammation (2022), 19 (1), 91CODEN: JNOEB3; ISSN:1742-2094. (BioMed Central Ltd.)
      The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (SPAK). Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacol. blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examd. Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was assocd. with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacol. blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. Stroke-induced patholol. stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.
    11. 11
      Chew, T. A.; Orlando, B. J.; Zhang, J.; Latorraca, N. R.; Wang, A.; Hollingsworth, S. A.; Chen, D.-H.; Dror, R. O.; Liao, M.; Feng, L. Structure and Mechanism of the Cation–Chloride Cotransporter NKCC1. Nature 2019, 572 (7770), 488– 492,  DOI: 10.1038/s41586-019-1438-2
      11
      Structure and mechanism of the cation-chloride cotransporter NKCC1
      Chew, Thomas A.; Orlando, Benjamin J.; Zhang, Jinru; Latorraca, Naomi R.; Wang, Amy; Hollingsworth, Scott A.; Chen, Dong-Hua; Dror, Ron O.; Liao, Maofu; Feng, Liang
      Nature (London, United Kingdom) (2019), 572 (7770), 488-492CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have crit. roles in regulating cell vol., controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we detd. the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiol. functions of CCCs and interpreting disease-related mutations.
    12. 12
      Moseng, M. A.; Su, C.-C.; Rios, K.; Cui, M.; Lyu, M.; Glaza, P.; Klenotic, P. A.; Delpire, E.; Yu, E. W. Inhibition Mechanism of NKCC1 Involves the Carboxyl Terminus and Long-Range Conformational Coupling. Sci. Adv. 2022, 8 (43), eabq0952  DOI: 10.1126/sciadv.abq0952
      There is no corresponding record for this reference.
    13. 13
      Yang, X.; Wang, Q.; Cao, E. Structure of the Human Cation–Chloride Cotransporter NKCC1 Determined by Single-Particle Electron Cryo-Microscopy. Nat. Commun. 2020, 11 (1), 1016,  DOI: 10.1038/s41467-020-14790-3
      13
      Structure of the human cation-chloride cotransporter NKCC1 determined by single-particle electron cryo-microscopy
      Yang, Xiaoyong; Wang, Qinzhe; Cao, Erhu
      Nature Communications (2020), 11 (1), 1016CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      The secondary active cation-chloride cotransporters (CCCs) utilize the existing Na+ and/or K+ gradients to move Cl- into or out of cells. NKCC1 is an intensively studied member of the CCC family and plays fundamental roles in regulating trans-epithelial ion movement, cell vol., chloride homeostasis and neuronal excitability. Here, we report a cryo-EM structure of human NKCC1 captured in a partially loaded, inward-open state. NKCC1 assembles into a dimer, with the first ten transmembrane (TM) helixes harboring the transport core and TM11-TM12 helixes lining the dimer interface. TM1 and TM6 helixes break α-helical geometry halfway across the lipid bilayer where ion binding sites are organized around these discontinuous regions. NKCC1 may harbor multiple extracellular entryways and intracellular exits, raising the possibility that K+, Na+, and Cl- ions may traverse along their own routes for translocation. NKCC1 structure provides a blueprint for further probing structure-function relationships of NKCC1 and other CCCs.
    14. 14
      Zhang, S.; Zhou, J.; Zhang, Y.; Liu, T.; Friedel, P.; Zhuo, W.; Somasekharan, S.; Roy, K.; Zhang, L.; Liu, Y.; Meng, X.; Deng, H.; Zeng, W.; Li, G.; Forbush, B.; Yang, M. The Structural Basis of Function and Regulation of Neuronal Cotransporters NKCC1 and KCC2. Commun. Biol. 2021, 4 (1), 226,  DOI: 10.1038/s42003-021-01750-w
      14
      The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2
      Zhang, Sensen; Zhou, Jun; Zhang, Yuebin; Liu, Tianya; Friedel, Perrine; Zhuo, Wei; Somasekharan, Suma; Roy, Kasturi; Zhang, Laixing; Liu, Yang; Meng, Xianbin; Deng, Haiteng; Zeng, Wenwen; Li, Guohui; Forbush, Biff; Yang, Maojun
      Communications Biology (2021), 4 (1), 226CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
      NKCC and KCC transporters mediate coupled transport of Na++K++Cl- and K++Cl- across the plasma membrane, thus regulating cell Cl- concn. and cell vol. and playing crit. roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational anal. and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.
    15. 15
      Zhao, Y.; Roy, K.; Vidossich, P.; Cancedda, L.; De Vivo, M.; Forbush, B.; Cao, E. Structural Basis for Inhibition of the Cation-Chloride Cotransporter NKCC1 by the Diuretic Drug Bumetanide. Nat. Commun. 2022, 13 (1), 2747,  DOI: 10.1038/s41467-022-30407-3
      15
      Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide
      Zhao, Yongxiang; Roy, Kasturi; Vidossich, Pietro; Cancedda, Laura; De Vivo, Marco; Forbush, Biff; Cao, Erhu
      Nature Communications (2022), 13 (1), 2747CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl- across cell membranes. NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons and NKCC2 is crit. to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with sepd. transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain assocn.
    16. 16
      Krishnamurthy, H.; Gouaux, E. X-Ray Structures of LeuT in Substrate-Free Outward-Open and Apo Inward-Open States. Nature 2012, 481 (7382), 469– 474,  DOI: 10.1038/nature10737
      16
      X-ray structures of LeuT in substrate-free outward-open and apo inward-open states
      Krishnamurthy, Harini; Gouaux, Eric
      Nature (London, United Kingdom) (2012), 481 (7382), 469-474CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Neurotransmitter sodium symporters are integral membrane proteins that remove chem. transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homolog, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.
    17. 17
      del Alamo, D.; Meiler, J.; Mchaourab, H. S. Principles of Alternating Access in LeuT-Fold Transporters: Commonalities and Divergences. J. Mol. Biol. 2022, 434 (19), 167746  DOI: 10.1016/j.jmb.2022.167746
      17
      Principles of Alternating Access in LeuT-fold Transporters: Commonalities and Divergences
      del Alamo, Diego; Meiler, Jens; Mchaourab, Hassane S.
      Journal of Molecular Biology (2022), 434 (19), 167746CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      A review. Found in all domains of life, transporters belonging to the LeuT-fold class mediate the import and exchange of hydrophilic and charged compds. such as amino acids, metals, and sugar mols. Nearly two decades of studies on the eponymous bacterial transporter LeuT have yielded a library of high-resoln. snapshots of its conformational cycle linked by soln.-state exptl. data obtained from multiple techniques. In parallel, its topol. was obsd. in symporters and antiporters characterized by a spectrum of substrate specificities and coupled to gradients of distinct ions. Here the authors review and compare mechanistic models of transport for LeuT, its well-studied homologs, as well as functionally distant members of the fold, emphasizing the commonalities and divergences in alternating access and the corresponding energy landscapes. The authors' integrated summary illustrates how fold conservation, a hallmark of the LeuT fold, coincides with divergent choreogs. of alternating access that nevertheless capitalize on recurrent structural motifs. In addn., it highlights the knowledge gap that hinders the leveraging of the current body of research into detailed mechanisms of transport for this important class of membrane proteins.
    18. 18
      Bonati, L.; Rizzi, V.; Parrinello, M. Data-Driven Collective Variables for Enhanced Sampling. J. Phys. Chem. Lett. 2020, 11 (8), 2998– 3004,  DOI: 10.1021/acs.jpclett.0c00535
      18
      Data-Driven Collective Variables for Enhanced Sampling
      Bonati, Luigi; Rizzi, Valerio; Parrinello, Michele
      Journal of Physical Chemistry Letters (2020), 11 (8), 2998-3004CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Designing an appropriate set of collective variables is crucial to the success of several enhanced sampling methods. Here we focus on how to obtain such variables from information limited to the metastable states. We characterize these states by a large set of descriptors and employ neural networks to compress this information in a lower-dimensional space, using Fisher's linear discriminant as an objective function to maximize the discriminative power of the network. We test this method on alanine dipeptide, using the nonlinearly separable data set composed by at. distances. We then study an intermol. aldol reaction characterized by a concerted mechanism. The resulting variables are able to promote sampling by drawing nonlinear paths in the phys. space connecting the fluctuations between metastable basins. Lastly, we interpret the behavior of the neural network by studying its relation to the phys. variables. Through the identification of its most relevant features, we are able to gain chem. insight into the process.
    19. 19
      Invernizzi, M.; Parrinello, M. Exploration vs Convergence Speed in Adaptive-Bias Enhanced Sampling. J. Chem. Theory Comput. 2022, 18 (6), 3988– 3996,  DOI: 10.1021/acs.jctc.2c00152
      19
      Exploration vs Convergence Speed in Adaptive-Bias Enhanced Sampling
      Invernizzi, Michele; Parrinello, Michele
      Journal of Chemical Theory and Computation (2022), 18 (6), 3988-3996CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      In adaptive-bias enhanced sampling methods, a bias potential is added to the system to drive transitions between metastable states. The bias potential is a function of a few collective variables and is gradually modified according to the underlying free energy surface. We show that when the collective variables are suboptimal, there is an exploration-convergence tradeoff, and one must choose between a quickly converging bias that will lead to fewer transitions or a slower to converge bias that can explore the phase space more efficiently but might require a much longer time to produce an accurate free energy est. The recently proposed on-the-fly probability enhanced sampling (OPES) method focuses on fast convergence, but there are cases where fast exploration is preferred instead. For this reason, we introduce a new variant of the OPES method that focuses on quickly escaping metastable states at the expense of convergence speed. We illustrate the benefits of this approach in prototypical systems and show that it outperforms the popular metadynamics method.
    20. 20
      Invernizzi, M.; Parrinello, M. Rethinking Metadynamics: From Bias Potentials to Probability Distributions. J. Phys. Chem. Lett. 2020, 11 (7), 2731– 2736,  DOI: 10.1021/acs.jpclett.0c00497
      20
      Rethinking Metadynamics: From Bias Potentials to Probability Distributions
      Invernizzi, Michele; Parrinello, Michele
      Journal of Physical Chemistry Letters (2020), 11 (7), 2731-2736CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Metadynamics is an enhanced sampling method of great popularity, based on the on-the-fly construction of a bias potential that is a function of a selected no. of collective variables. We propose here a change in perspective that shifts the focus from the bias to the probability distribution reconstruction while retaining some of the key characteristics of metadynamics, such as flexible on-the-fly adjustments to the free energy est. The result is an enhanced sampling method that presents a drastic improvement in convergence speed, esp. when dealing with suboptimal and/or multidimensional sets of collective variables. The method is esp. robust and easy to use and in fact requires only a few simple parameters to be set, and it has a straightforward reweighting scheme to recover the statistics of the unbiased ensemble. Furthermore, it gives more control of the desired exploration of the phase space since the deposited bias is not allowed to grow indefinitely and it does not push the simulation to uninteresting high free energy regions. We demonstrate the performance of the method in a no. of representative examples.
    21. 21
      Yamashita, A.; Singh, S. K.; Kawate, T.; Jin, Y.; Gouaux, E. Crystal Structure of a Bacterial Homologue of Na+/Cl--Dependent Neurotransmitter Transporters. Nature 2005, 437 (7056), 215– 223,  DOI: 10.1038/nature03978
      21
      Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters
      Yamashita, Atsuko; Singh, Satinder K.; Kawate, Toshimitsu; Jin, Yan; Gouaux, Eric
      Nature (London, United Kingdom) (2005), 437 (7056), 215-223CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Na+/Cl--dependent transporters terminate synaptic transmission by using electrochem. gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compds., and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homolog of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helixes, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.
    22. 22
      Forrest, L. R.; Rudnick, G. The Rocking Bundle: A Mechanism for Ion-Coupled Solute Flux by Symmetrical Transporters. Physiology 2009, 24 (6), 377– 386,  DOI: 10.1152/physiol.00030.2009
      22
      The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters
      Forrest, Lucy R.; Rudnick, Gary
      Physiology (2009), 24 (Dec.), 377-386CODEN: PHYSCI; ISSN:1548-9213. (International Union of Physiological Sciences)
      A review. Crystal structures of the bacterial amino acid transporter LeuT have provided the basis for understanding the conformational changes assocd. with substrate translocation by a multitude of transport proteins with the same fold. Biochem. and modeling studies led to a "rocking bundle" mechanism for LeuT that was validated by subsequent transporter structures. These advances suggest how coupled solute transport might be defined by the internal symmetry of proteins contg. inverted structural repeats.
    23. 23
      Masrati, G.; Mondal, R.; Rimon, A.; Kessel, A.; Padan, E.; Lindahl, E.; Ben-Tal, N. An Angular Motion of a Conserved Four-Helix Bundle Facilitates Alternating Access Transport in the TtNapA and EcNhaA Transporters. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (50), 31850– 31860,  DOI: 10.1073/pnas.2002710117
      23
      An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters
      Masrati, Gal; Mondal, Ramakanta; Rimon, Abraham; Kessel, Amit; Padan, Etana; Lindahl, Erik; Ben-Tal, Nir
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (50), 31850-31860CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, resp. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using crosslinking expts.
    24. 24
      Borgogno, M.; Savardi, A.; Manigrasso, J.; Turci, A.; Portioli, C.; Ottonello, G.; Bertozzi, S. M.; Armirotti, A.; Contestabile, A.; Cancedda, L.; De Vivo, M. Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome. J. Med. Chem. 2021, 64 (14), 10203– 10229,  DOI: 10.1021/acs.jmedchem.1c00603
      24
      Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome
      Borgogno, Marco; Savardi, Annalisa; Manigrasso, Jacopo; Turci, Alessandra; Portioli, Corinne; Ottonello, Giuliana; Bertozzi, Sine Mandrup; Armirotti, Andrea; Contestabile, Andrea; Cancedda, Laura; De Vivo, Marco
      Journal of Medicinal Chemistry (2021), 64 (14), 10203-10229CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Intracellular chloride concn. [Cl-]i is defective in several neurol. disorders. In neurons, [Cl-]i is mainly regulated by the action of the Na+-K+-Cl- importer NKCC1 and the K+-Cl- exporter KCC2. Recently, we have reported the discovery of ARN23746(I) as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chem. class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivs. In particular, we present compd. 40 (ARN24092)(II) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compds. for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.
    25. 25
      Savardi, A.; Borgogno, M.; Narducci, R.; La Sala, G.; Ortega, J. A.; Summa, M.; Armirotti, A.; Bertorelli, R.; Contestabile, A.; De Vivo, M.; Cancedda, L. Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders. Chem. 2020, 6 (8), 2073– 2096,  DOI: 10.1016/j.chempr.2020.06.017
      25
      Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders
      Savardi, Annalisa; Borgogno, Marco; Narducci, Roberto; La Sala, Giuseppina; Ortega, Jose Antonio; Summa, Maria; Armirotti, Andrea; Bertorelli, Rosalia; Contestabile, Andrea; De Vivo, Marco; Cancedda, Laura
      Chem (2020), 6 (8), 2073-2096CODEN: CHEMVE; ISSN:2451-9294. (Cell Press)
      Aberrant expression ratio of Cl- transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clin. trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl- transporter NKCC2, creating crit. drug compliance issues and health concerns. Here, we report the discovery of a new chem. class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiol. intracellular Cl- in murine Down syndrome neuronal cultures, has excellent soly. and metabolic stability, and displays no issues with off-target activity in vitro. ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclin./manufg. studies toward the first sustainable therapeutics for the neurol. conditions characterized by impaired Cl- homeostasis.
    26. 26
      Savardi, A.; Patricelli Malizia, A.; De Vivo, M.; Cancedda, L.; Borgogno, M. Preclinical Development of the Na-K-2Cl Co-Transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders. ACS Pharmacol. Transl. Sci. 2023, 6 (1), 1– 11,  DOI: 10.1021/acsptsci.2c00197
      26
      Preclinical Development of the Na-K-2Cl Co-transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders
      Savardi, Annalisa; Patricelli Malizia, Andrea; De Vivo, Marco; Cancedda, Laura; Borgogno, Marco
      ACS Pharmacology & Translational Science (2023), 6 (1), 1-11CODEN: APTSFN; ISSN:2575-9108. (American Chemical Society)
      Alterations in the expression of the Cl- importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl co-transporter 2 (KCC2) lead to impaired intracellular chloride concn. in neurons and imbalanced excitation/inhibition in the brain. These alterations have been obsd. in several neurol. disorders (e.g., Down syndrome and autism). Recently, we have reported the discovery of the selective NKCC1 inhibitor "compd. ARN23746" for the treatment of Down syndrome and autism in mouse models. Here, we report on an extensive preclin. characterization of ARN23746 toward its development as a clin. candidate. ARN23746 shows an overall excellent metab. profile and good brain penetration. Moreover, ARN23746 is effective in rescuing cognitive impairment in Down syndrome mice upon per os administration, in line with oral treatment of neurodevelopmental disorders. Notably, ARN23746 does not present signs of toxicity or diuresis even if administered up to 50 times the ED. These results further support ARN23746 as a solid candidate for clin. trial-enabling studies.
    27. 27
      Chew, T. A.; Zhang, J.; Feng, L. High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters. J. Mol. Biol. 2021, 433 (16), 167056  DOI: 10.1016/j.jmb.2021.167056
      27
      High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters
      Chew, Thomas A.; Zhang, Jinru; Feng, Liang
      Journal of Molecular Biology (2021), 433 (16), 167056CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      A review. Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl- with K+ and/or Na+ in an electroneutral manner. They play important roles in myriad fundamental physiol. processes--from cell vol. regulation to transepithelial solute transport and intracellular ion homeostasis--and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacol. of these transporters, there have been several breakthroughs in the structural detn. of CCC transporters. The insights provided by these new structures for the Na+/K+/Cl- cotransporter NKCC1 and the K+/Cl- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their mol. basis and transport function. This focused discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.
    28. 28
      Lytle, C.; McManus, T. J.; Haas, M. A Model of Na-K-2Cl Cotransport Based on Ordered Ion Binding and Glide Symmetry. Am. J. Physiol.-Cell Physiol. 1998, 274 (2), C299– C309,  DOI: 10.1152/ajpcell.1998.274.2.C299
      There is no corresponding record for this reference.
    29. 29
      Delpire, E.; Gagnon, K. B. Na + -K + −2Cl – Cotransporter (NKCC) Physiological Function in Nonpolarized Cells and Transporting Epithelia. In Comprehensive Physiology; Terjung, R., Ed.; Wiley, 2018; pp. 871– 901.
      There is no corresponding record for this reference.
    30. 30
      Shimamura, T.; Weyand, S.; Beckstein, O.; Rutherford, N. G.; Hadden, J. M.; Sharples, D.; Sansom, M. S. P.; Iwata, S.; Henderson, P. J. F.; Cameron, A. D. Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1. Science 2010, 328 (5977), 470– 473,  DOI: 10.1126/science.1186303
      30
      Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1
      Shimamura, Tatsuro; Weyand, Simone; Beckstein, Oliver; Rutherford, Nicholas G.; Hadden, Jonathan M.; Sharples, David; Sansom, Mark S. P.; Iwata, So; Henderson, Peter J. F.; Cameron, Alexander D.
      Science (Washington, DC, United States) (2010), 328 (5977), 470-473CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens is comprised of a five-helix inverted repeat, a conformation widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resoln., which complements previously described structures of Mhp1 in outward-facing and occluded states. From analyses of the three structures and mol. dynamics (MD) simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helixes 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
    31. 31
      Hamann, S.; Herrera-Perez, J. J.; Zeuthen, T.; Alvarez-Leefmans, F. J. Cotransport of Water by the Na + -K + −2Cl – Cotransporter NKCC1 in Mammalian Epithelial Cells: Cotransport of Water by NKCC1. J. Physiol. 2010, 588 (21), 4089– 4101,  DOI: 10.1113/jphysiol.2010.194738
      31
      Cotransport of water by the Na+-K+-2Cl- cotransporter NKCC1 in mammalian epithelial cells
      Hamann, Steffen; Herrera-Perez, Jose J.; Zeuthen, Thomas; Alvarez-Leefmans, Francisco J.
      Journal of Physiology (Oxford, United Kingdom) (2010), 588 (21), 4089-4101CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
      Water transport by the Na+-K+-2Cl- cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl- fluxes mediated by NKCC1 was inferred from changes in cell water vol., monitored by intracellular self-quenching of the fluorescent dye calcein. Isosmotic removal of external Cl- or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μM). When returned to the control soln. there was a rapid water influx that required the simultaneous presence of external Na+ and Cl-. The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl-] satd. in a sigmoidal fashion with a Km of 60 mM, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mM. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their vol. independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and sym. cells.
    32. 32
      Zeuthen, T.; MacAulay, N. Cotransport of Water by Na + -K + −2Cl – Cotransporters Expressed in Xenopus Oocytes: NKCC1 versus NKCC2: Water Transport in NKCC. J. Physiol. 2012, 590 (5), 1139– 1154,  DOI: 10.1113/jphysiol.2011.226316
      32
      Cotransport of water by Na+-K+-2Cl- cotransporters expressed in Xenopus oocytes: NKCC1 versus NKCC2
      Zeuthen, Thomas; MacAulay, Nanna
      Journal of Physiology (Oxford, United Kingdom) (2012), 590 (5), 1139-1154CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
      The NKCC1 and NKCC2 isoforms of the mammalian Na+-K+-2Cl- cotransporter were expressed in Xenopus oocytes and the relation between external ion concn. and water fluxes detd. Water fluxes were detd. from changes in the oocytes vol. and ion fluxes from 86Rb+ uptake. Isotonic increases in external K+ concn. elicited abrupt inward water fluxes in NKCC1; the K+ dependence obeyed one-site kinetics with a K0.5 of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temp. dependence and could proceed uphill against an osmotic gradient of 20 mosmol L-1. A comparison between ion and water fluxes indicates that 460 water mols. are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes. Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the notion of interaction between ions and water in NKCC1 and allows for an est. of around 600 water mols. transported per turnover of the protein. Osmotic gradients did not induce water transport in NKCC2. We conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfills its role in the kidney of transporting ions but not water. The different behavior of NKCC1 and NKCC2 is discussed on the basis of recent mol. models based on studies of structural and mol. dynamics.
    33. 33
      Sadegh, C.; Xu, H.; Sutin, J.; Fatou, B.; Gupta, S.; Pragana, A.; Taylor, M.; Kalugin, P. N.; Zawadzki, M. E.; Alturkistani, O.; Shipley, F. B.; Dani, N.; Fame, R. M.; Wurie, Z.; Talati, P.; Schleicher, R. L.; Klein, E. M.; Zhang, Y.; Holtzman, M. J.; Moore, C. I.; Lin, P.-Y.; Patel, A. B.; Warf, B. C.; Kimberly, W. T.; Steen, H.; Andermann, M. L.; Lehtinen, M. K. Choroid Plexus-Targeted NKCC1 Overexpression to Treat Post-Hemorrhagic Hydrocephalus. Neuron 2023, 111 (10), 1591– 1608,  DOI: 10.1016/j.neuron.2023.02.020
      33
      Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus
      Sadegh, Cameron; Xu, Huixin; Sutin, Jason; Fatou, Benoit; Gupta, Suhasini; Pragana, Aja; Taylor, Milo; Kalugin, Peter N.; Zawadzki, Miriam E.; Alturkistani, Osama; Shipley, Frederick B.; Dani, Neil; Fame, Ryann M.; Wurie, Zainab; Talati, Pratik; Schleicher, Riana L.; Klein, Eric M.; Zhang, Yong; Holtzman, Michael J.; Moore, Christopher I.; Lin, Pei-Yi; Patel, Aman B.; Warf, Benjamin C.; Kimberly, W. Taylor; Steen, Hanno; Andermann, Mark L.; Lehtinen, Maria K.
      Neuron (2023), 111 (10), 1591-1608.e4CODEN: NERNET; ISSN:0896-6273. (Cell Press)
      Post-hemorrhagic hydrocephalus (PHH) refers to a life-threatening accumulation of cerebrospinal fluid (CSF) that occurs following intraventricular hemorrhage (IVH). An incomplete understanding of this variably progressive condition has hampered the development of new therapies beyond serial neurosurgical interventions. Here, we show a key role for the bidirectional Na-K-Cl cotransporter, NKCC1, in the choroid plexus (ChP) to mitigate PHH. Mimicking IVH with intraventricular blood led to increased CSF [K+] and triggered cytosolic calcium activity in ChP epithelial cells, which was followed by NKCC1 activation. ChP-targeted adeno-assocd. viral (AAV)-NKCC1 prevented blood-induced ventriculomegaly and led to persistently increased CSF clearance capacity. These data demonstrate that intraventricular blood triggered a trans-choroidal, NKCC1-dependent CSF clearance mechanism. Inactive, phosphodeficient AAV-NKCC1-NT51 failed to mitigate ventriculomegaly. Excessive CSF [K+] fluctuations correlated with permanent shunting outcome in humans following hemorrhagic stroke, suggesting targeted gene therapy as a potential treatment to mitigate intracranial fluid accumulation following hemorrhage.
    34. 34
      Steffensen, A. B.; Oernbo, E. K.; Stoica, A.; Gerkau, N. J.; Barbuskaite, D.; Tritsaris, K.; Rose, C. R.; MacAulay, N. Cotransporter-Mediated Water Transport Underlying Cerebrospinal Fluid Formation. Nat. Commun. 2018, 9 (1), 2167,  DOI: 10.1038/s41467-018-04677-9
      34
      Cotransporter-mediated water transport underlying cerebrospinal fluid formation
      Steffensen Annette B; Oernbo Eva K; Stoica Anca; MacAulay Nanna; Gerkau Niklas J; Rose Christine R; Barbuskaite Dagne; Tritsaris Katerina
      Nature communications (2018), 9 (1), 2167 ISSN:.
      Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na(+)/K(+)/2Cl(-) cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.
    35. 35
      Li, J.; Shaikh, S. A.; Enkavi, G.; Wen, P.-C.; Huang, Z.; Tajkhorshid, E. Transient Formation of Water-Conducting States in Membrane Transporters. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (19), 7696– 7701,  DOI: 10.1073/pnas.1218986110
      35
      Transient formation of water-conducting states in membrane transporters
      Li, Jing; Shaikh, Saher A.; Enkavi, Giray; Wen, Po-Chao; Huang, Zhijian; Tajkhorshid, Emad
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (19), 7696-7701, S7696/1-S7696/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane - a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mech. coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equil. mol. dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying mol. mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the obsd. water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.
    36. 36
      Okazaki, K.; Wöhlert, D.; Warnau, J.; Jung, H.; Yildiz, Ö.; Kühlbrandt, W.; Hummer, G. Mechanism of the Electroneutral Sodium/Proton Antiporter PaNhaP from Transition-Path Shooting. Nat. Commun. 2019, 10 (1), 1742,  DOI: 10.1038/s41467-019-09739-0
      36
      Mechanism of the electroneutral sodium/proton antiporter PaNhaP from transition-path shooting
      Okazaki Kei-Ichi; Okazaki Kei-Ichi; Warnau Judith; Jung Hendrik; Hummer Gerhard; Wohlert David; Yildiz Ozkan; Kuhlbrandt Werner; Hummer Gerhard
      Nature communications (2019), 10 (1), 1742 ISSN:.
      Na(+)/H(+) antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na(+)/H(+) antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na(+)/H(+) exchanger NHE1, which is an important drug target. Here we resolve the Na(+) and H(+) transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.
    37. 37
      Monette, M. Y.; Somasekharan, S.; Forbush, B. Molecular Motions Involved in Na-K-Cl Cotransporter-Mediated Ion Transport and Transporter Activation Revealed by Internal Cross-Linking between Transmembrane Domains 10 and 11/12. J. Biol. Chem. 2014, 289 (11), 7569– 7579,  DOI: 10.1074/jbc.M113.542258
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      Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Cross-linking between Transmembrane Domains 10 and 11/12
      Monette, Michelle Y.; Somasekharan, Suma; Forbush, Biff
      Journal of Biological Chemistry (2014), 289 (11), 7569-7579CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      We examd. the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homol. models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function; indeed, evidence for cross-link formation with low micromolar concns. of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 addnl. tested pairs. Inhibition of transport was obsd. with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu2+ inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met382 of TM3 are involved in a Cu2+-binding site. Surprisingly, crosslinking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) crosslinking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by crosslinking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.
    38. 38
      McNeill, A.; Iovino, E.; Mansard, L.; Vache, C.; Baux, D.; Bedoukian, E.; Cox, H.; Dean, J.; Goudie, D.; Kumar, A.; Newbury-Ecob, R.; Fallerini, C.; Renieri, A.; Lopergolo, D.; Mari, F.; Blanchet, C.; Willems, M.; Roux, A.-F.; Pippucci, T.; Delpire, E. SLC12A2 Variants Cause a Neurodevelopmental Disorder or Cochleovestibular Defect. Brain 2020, 143 (8), 2380– 2387,  DOI: 10.1093/brain/awaa176
      38
      SLC12A2 variants cause a neurodevelopmental disorder or cochleovestibular defect
      McNeill Alisdair; McNeill Alisdair; McNeill Alisdair; Iovino Emanuela; Mansard Luke; Vache Christel; Baux David; Roux Anne-Francoise; Bedoukian Emma; Cox Helen; Dean John; Goudie David; Kumar Ajith; Newbury-Ecob Ruth; Fallerini Chiara; Renieri Alessandra; Lopergolo Diego; Mari Francesca; Fallerini Chiara; Renieri Alessandra; Lopergolo Diego; Mari Francesca; Blanchet Catherine; Willems Marjolaine; Pippucci Tommaso; Delpire Eric
      Brain : a journal of neurology (2020), 143 (8), 2380-2387 ISSN:.
      The SLC12 gene family consists of SLC12A1-SLC12A9, encoding electroneutral cation-coupled chloride co-transporters. SCL12A2 has been shown to play a role in corticogenesis and therefore represents a strong candidate neurodevelopmental disorder gene. Through trio exome sequencing we identified de novo mutations in SLC12A2 in six children with neurodevelopmental disorders. All had developmental delay or intellectual disability ranging from mild to severe. Two had sensorineural deafness. We also identified SLC12A2 variants in three individuals with non-syndromic bilateral sensorineural hearing loss and vestibular areflexia. The SLC12A2 de novo mutation rate was demonstrated to be significantly elevated in the deciphering developmental disorders cohort. All tested variants were shown to reduce co-transporter function in Xenopus laevis oocytes. Analysis of SLC12A2 expression in foetal brain at 16-18 weeks post-conception revealed high expression in radial glial cells, compatible with a role in neurogenesis. Gene co-expression analysis in cells robustly expressing SLC12A2 at 16-18 weeks post-conception identified a transcriptomic programme associated with active neurogenesis. We identify SLC12A2 de novo mutations as the cause of a novel neurodevelopmental disorder and bilateral non-syndromic sensorineural hearing loss and provide further data supporting a role for this gene in human neurodevelopment.
    39. 39
      Janoš, P.; Magistrato, A. All-Atom Simulations Uncover the Molecular Terms of the NKCC1 Transport Mechanism. J. Chem. Inf. Model. 2021, 61 (7), 3649– 3658,  DOI: 10.1021/acs.jcim.1c00551
      39
      All-Atom Simulations Uncover the Molecular Terms of the NKCC1 Transport Mechanism
      Janos, Pavel; Magistrato, Alessandra
      Journal of Chemical Information and Modeling (2021), 61 (7), 3649-3658CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      The secondary-active Na-K-Cl cotransporter 1 (NKCC1), member of the cation-chloride cotransporter (CCC) family, ensures the electroneutral movement of Cl-, Na+, and K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell vol., handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, and cancer). Building on the newly resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ion transport, assessing the order and the mol. basis of its interdependent ion translocation. The authors' outcomes strikingly advance the understanding of the physiol. mechanism of CCCs and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both neg. and pos. charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis.
    40. 40
      Portioli, C.; Ruiz Munevar, M. J.; De Vivo, M.; Cancedda, L. Cation-Coupled Chloride Cotransporters: Chemical Insights and Disease Implications. Trends Chem. 2021, 3 (10), 832– 849,  DOI: 10.1016/j.trechm.2021.05.004
      40
      Cation-coupled chloride cotransporters: chemical insights and disease implications
      Portioli, Corinne; Ruiz Munevar, Manuel Jose; De Vivo, Marco; Cancedda, Laura
      Trends in Chemistry (2021), 3 (10), 832-849CODEN: TCRHBQ; ISSN:2589-5974. (Cell Press)
      A review. Cation-coupled chloride cotransporters (CCCs) modulate the transport of sodium and/or potassium cations coupled with chloride anions across the cell membrane. CCCs thus help regulate intracellular ionic concn. and consequent cell vol. homeostasis. This has been largely exploited in the past to develop diuretic drugs that act on CCCs expressed in the kidney. However, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. Here, we examine the structure-function relationship of CCCs. By linking recent high-resoln. cryogenic electron microscopy (cryo-EM) data with older biochem./functional studies on CCCs, we discuss the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies.
    41. 41
      Zhao, Y.; Shen, J.; Wang, Q.; Ruiz Munevar, M. J.; Vidossich, P.; De Vivo, M.; Zhou, M.; Cao, E. Structure of the Human Cation–Chloride Cotransport KCC1 in an Outward-Open State. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (27), e2109083119  DOI: 10.1073/pnas.2109083119
      41
      Structure of the human cation-chloride cotransport KCC1 in an outward-open state
      Zhao, Yongxiang; Shen, Jiemin; Wang, Qinzhe; Munevar, Manuel Jose Ruiz; Vidossich, Pietro; De Vivo, Marco; Zhou, Ming; Cao, Erhu
      Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (27), e2109083119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell vol. homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concn., and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helixes. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small mol. compds.
    42. 42
      Saitsu, H.; Watanabe, M.; Akita, T.; Ohba, C.; Sugai, K.; Ong, W. P.; Shiraishi, H.; Yuasa, S.; Matsumoto, H.; Beng, K. T.; Saitoh, S.; Miyatake, S.; Nakashima, M.; Miyake, N.; Kato, M.; Fukuda, A.; Matsumoto, N. Impaired Neuronal KCC2 Function by Biallelic SLC12A5Mutations in Migrating Focal Seizures and Severe Developmental Delay. Sci. Rep. 2016, 6 (1), 30072,  DOI: 10.1038/srep30072
      42
      Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay
      Saitsu, Hirotomo; Watanabe, Miho; Akita, Tenpei; Ohba, Chihiro; Sugai, Kenji; Ong, Winnie Peitee; Shiraishi, Hideaki; Yuasa, Shota; Matsumoto, Hiroshi; Beng, Khoo Teik; Saitoh, Shinji; Miyatake, Satoko; Nakashima, Mitsuko; Miyake, Noriko; Kato, Mitsuhiro; Fukuda, Atsuo; Matsumoto, Naomichi
      Scientific Reports (2016), 6 (), 30072CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compd. heterozygous SLC12A5 (encoding the neuronal K+-Cl- co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compd. heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp anal. demonstrated strongly suppressed Cl- extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl- level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl- extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS.
    43. 43
      Uyanik, G.; Elcioglu, N.; Penzien, J.; Gross, C.; Yilmaz, Y.; Olmez, A.; Demir, E.; Wahl, D.; Scheglmann, K.; Winner, B.; Bogdahn, U.; Topaloglu, H.; Hehr, U.; Winkler, J. Novel Truncating and Missense Mutations of the KCC3 Gene Associated with Andermann Syndrome. Neurology 2006, 67 (7), 1044,  DOI: 10.1212/01.wnl.0000250608.09509.ed
      There is no corresponding record for this reference.
    44. 44
      Olsson, M. H. M.; So̷ndergaard, C. R.; Rostkowski, M.; Jensen, J. H. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p Ka Predictions. J. Chem. Theory Comput. 2011, 7 (2), 525– 537,  DOI: 10.1021/ct100578z
      44
      PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions
      Olsson, Mats H. M.; Sondergaard, Chresten R.; Rostkowski, Michal; Jensen, Jan H.
      Journal of Chemical Theory and Computation (2011), 7 (2), 525-537CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      The authors have revised the rules and parameters for one of the most commonly used empirical pKa predictors, PROPKA, based on better phys. description of the desolvation and dielec. response for the protein. The authors have introduced a new and consistent approach to interpolate the description between the previously distinct classifications into internal and surface residues, which otherwise is found to give rise to an erratic and discontinuous behavior. Since the goal of this study is to lay out the framework and validate the concept, it focuses on Asp and Glu residues where the protein pKa values and structures are assumed to be more reliable. The new and improved implementation is evaluated and discussed; it is found to agree better with expt. than the previous implementation (in parentheses): rmsd = 0.79 (0.91) for Asp and Glu, 0.75 (0.97) for Tyr, 0.65 (0.72) for Lys, and 1.00 (1.37) for His residues. The most significant advance, however, is in reducing the no. of outliers and removing unreasonable sensitivity to small structural changes that arise from classifying residues as either internal or surface.
    45. 45
      Schott-Verdugo, S.; Gohlke, H. PACKMOL-Memgen: A Simple-To-Use, Generalized Workflow for Membrane-Protein–Lipid-Bilayer System Building. J. Chem. Inf. Model. 2019, 59 (6), 2522– 2528,  DOI: 10.1021/acs.jcim.9b00269
      45
      PACKMOL-Memgen: A Simple-To-Use, Generalized Workflow for Membrane-Protein-Lipid-Bilayer System Building
      Schott-Verdugo, Stephan; Gohlke, Holger
      Journal of Chemical Information and Modeling (2019), 59 (6), 2522-2528CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      We present PACKMOL-Memgen, a simple-to-use, generalized workflow for automated building of membrane-protein-lipid-bilayer systems based on open-source tools including Packmol, memembed, pdbremix, and AmberTools. Compared with web-interface-based related tools, PACKMOL-Memgen allows setup of multiple configurations of a system in a user-friendly and efficient manner within minutes. The generated systems are well-packed and thus well-suited as starting configurations in MD simulations under periodic boundary conditions, requiring only moderate equilibration times. PACKMOL-Memgen is distributed with AmberTools and runs on most computing platforms, and its output can also be used for CHARMM or adapted to other mol.-simulation packages.
    46. 46
      Le Grand, S.; Götz, A. W.; Walker, R. C. SPFP: Speed without Compromise─A Mixed Precision Model for GPU Accelerated Molecular Dynamics Simulations. Comput. Phys. Commun. 2013, 184 (2), 374– 380,  DOI: 10.1016/j.cpc.2012.09.022
      46
      SPFP: Speed without compromise-A mixed precision model for GPU accelerated molecular dynamics simulations
      Le Grand, Scott; Gotz, Andreas W.; Walker, Ross C.
      Computer Physics Communications (2013), 184 (2), 374-380CODEN: CPHCBZ; ISSN:0010-4655. (Elsevier B.V.)
      A new precision model is proposed for the acceleration of all-atom classical mol. dynamics (MD) simulations on graphics processing units (GPUs). This precision model replaces double precision arithmetic with fixed point integer arithmetic for the accumulation of force components as compared to a previously introduced model that uses mixed single/double precision arithmetic. This significantly boosts performance on modern GPU hardware without sacrificing numerical accuracy. We present an implementation for NVIDIA GPUs of both generalized Born implicit solvent simulations as well as explicit solvent simulations using the particle mesh Ewald (PME) algorithm for long-range electrostatics using this precision model. Tests demonstrate both the performance of this implementation as well as its numerical stability for const. energy and const. temp. biomol. MD as compared to a double precision CPU implementation and double and mixed single/double precision GPU implementations.
    47. 47
      Salomon-Ferrer, R.; Case, D. A.; Walker, R. C. An Overview of the Amber Biomolecular Simulation Package: Amber Biomolecular Simulation Package. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2013, 3 (2), 198– 210,  DOI: 10.1002/wcms.1121
      47
      An overview of the amber biomolecular simulation package
      Salomon-Ferrer, Romelia; Case, David A.; Walker, Ross C.
      Wiley Interdisciplinary Reviews: Computational Molecular Science (2013), 3 (2), 198-210CODEN: WIRCAH; ISSN:1759-0884. (Wiley-Blackwell)
      A review. Mol. dynamics (MD) allows the study of biol. and chem. systems at the atomistic level on timescales from femtoseconds to milliseconds. It complements expt. while also offering a way to follow processes difficult to discern with exptl. techniques. Numerous software packages exist for conducting MD simulations of which one of the widest used is termed Amber. Here, we outline the most recent developments, since version 9 was released in Apr. 2006, of the Amber and AmberTools MD software packages, referred to here as simply the Amber package. The latest release represents six years of continued development, since version 9, by multiple research groups and the culmination of over 33 years of work beginning with the first version in 1979. The latest release of the Amber package, version 12 released in Apr. 2012, includes a substantial no. of important developments in both the scientific and computer science arenas. We present here a condensed vision of what Amber currently supports and where things are likely to head over the coming years. Figure 1 shows the performance in ns/day of the Amber package version 12 on a single-core AMD FX-8120 8-Core 3.6GHz CPU, the Cray XT5 system, and a single GPU GTX680.
    48. 48
      Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
      48
      ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
      Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
      Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
    49. 49
      Dickson, C. J.; Madej, B. D.; Skjevik, Å. A.; Betz, R. M.; Teigen, K.; Gould, I. R.; Walker, R. C. Lipid14: The Amber Lipid Force Field. J. Chem. Theory Comput. 2014, 10 (2), 865– 879,  DOI: 10.1021/ct4010307
      49
      Lipid14: The Amber Lipid Force Field
      Dickson, Callum J.; Madej, Benjamin D.; Skjevik, Age A.; Betz, Robin M.; Teigen, Knut; Gould, Ian R.; Walker, Ross C.
      Journal of Chemical Theory and Computation (2014), 10 (2), 865-879CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      The AMBER lipid force field has been updated to create Lipid14, allowing tensionless simulation of a no. of lipid types with the AMBER MD package. The modular nature of this force field allows numerous combinations of head and tail groups to create different lipid types, enabling the easy insertion of new lipid species. The Lennard-Jones and torsion parameters of both the head and tail groups have been revised and updated partial charges calcd. The force field has been validated by simulating bilayers of six different lipid types for a total of 0.5 μs each without applying a surface tension; with favorable comparison to expt. for properties such as area per lipid, vol. per lipid, bilayer thickness, NMR order parameters, scattering data, and lipid lateral diffusion. As the derivation of this force field is consistent with the AMBER development philosophy, Lipid14 is compatible with the AMBER protein, nucleic acid, carbohydrate, and small mol. force fields.
    50. 50
      Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79 (2), 926– 935,  DOI: 10.1063/1.445869
      50
      Comparison of simple potential functions for simulating liquid water
      Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.; Impey, Roger W.; Klein, Michael L.
      Journal of Chemical Physics (1983), 79 (2), 926-35CODEN: JCPSA6; ISSN:0021-9606.
      Classical Monte Carlo simulations were carried out for liq. H2O in the NPT ensemble at 25° and 1 atm using 6 of the simpler intermol. potential functions for the dimer. Comparisons were made with exptl. thermodn. and structural data including the neutron diffraction results of Thiessen and Narten (1982). The computed densities and potential energies agree with expt. except for the original Bernal-Fowler model, which yields an 18% overest. of the d. and poor structural results. The discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations. Comparisons were made for the self-diffusion coeffs. obtained from mol. dynamics simulations.
    51. 51
      Joung, I. S.; Cheatham, T. E. Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations. J. Phys. Chem. B 2008, 112 (30), 9020– 9041,  DOI: 10.1021/jp8001614
      51
      Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations
      Joung, In Suk; Cheatham, Thomas E.
      Journal of Physical Chemistry B (2008), 112 (30), 9020-9041CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      Alkali (Li+, Na+, K+, Rb+, and Cs+) and halide (F-, Cl-, Br-, and I-) ions play an important role in many biol. phenomena, roles that range from stabilization of biomol. structure, to influence on biomol. dynamics, to key physiol. influence on homeostasis and signaling. To properly model ionic interaction and stability in atomistic simulations of biomol. structure, dynamics, folding, catalysis, and function, an accurate model or representation of the monovalent ions is critically necessary. A good model needs to simultaneously reproduce many properties of ions, including their structure, dynamics, solvation, and moreover both the interactions of these ions with each other in the crystal and in soln. and the interactions of ions with other mols. At present, the best force fields for biomols. employ a simple additive, nonpolarizable, and pairwise potential for at. interaction. In this work, the authors describe their efforts to build better models of the monovalent ions within the pairwise Coulombic and 6-12 Lennard-Jones framework, where the models are tuned to balance crystal and soln. properties in Ewald simulations with specific choices of well-known water models. Although it has been clearly demonstrated that truly accurate treatments of ions will require inclusion of nonadditivity and polarizability (particularly with the anions) and ultimately even a quantum mech. treatment, the authors' goal was to simply push the limits of the additive treatments to see if a balanced model could be created. The applied methodol. is general and can be extended to other ions and to polarizable force-field models. The authors' starting point centered on observations from long simulations of biomols. in salt soln. with the AMBER force fields where salt crystals formed well below their soly. limit. The likely cause of the artifact in the AMBER parameters relates to the naive mixing of the Smith and Dang chloride parameters with AMBER-adapted Aqvist cation parameters. To provide a more appropriate balance, the authors reoptimized the parameters of the Lennard-Jones potential for the ions and specific choices of water models. To validate and optimize the parameters, the authors calcd. hydration free energies of the solvated ions and also lattice energies (LE) and lattice consts. (LC) of alkali halide salt crystals. This is the first effort that systematically scans across the Lennard-Jones space (well depth and radius) while balancing ion properties like LE and LC across all pair combinations of the alkali ions and halide ions. The optimization across the entire monovalent series avoids systematic deviations. The ion parameters developed, optimized, and characterized were targeted for use with some of the most commonly used rigid and nonpolarizable water models, specifically TIP3P, TIP4PEW, and SPC/E. In addn. to well reproducing the soln. and crystal properties, the new ion parameters well reproduce binding energies of the ions to water and the radii of the first hydration shells.
    52. 52
      Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An N ·log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98 (12), 10089– 10092,  DOI: 10.1063/1.464397
      52
      Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems
      Darden, Tom; York, Darrin; Pedersen, Lee
      Journal of Chemical Physics (1993), 98 (12), 10089-92CODEN: JCPSA6; ISSN:0021-9606.
      An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolution using fast Fourier transforms. Timings and accuracies are presented for three large cryst. ionic systems.
    53. 53
      Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23 (3), 327– 341,  DOI: 10.1016/0021-9991(77)90098-5
      53
      Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes
      Ryckaert, Jean Paul; Ciccotti, Giovanni; Berendsen, Herman J. C.
      Journal of Computational Physics (1977), 23 (3), 327-41CODEN: JCTPAH; ISSN:0021-9991.
      A numerical algorithm integrating the 3N Cartesian equation of motion of a system of N points subject to holonomic constraints is applied to mol. dynamics simulation of a liq. of 64 butane mols.
    54. 54
      Ansari, N.; Rizzi, V.; Parrinello, M. Water Regulates the Residence Time of Benzamidine in Trypsin. Nat. Commun. 2022, 13 (1), 5438,  DOI: 10.1038/s41467-022-33104-3
      54
      Water regulates the residence time of Benzamidine in Trypsin
      Ansari, Narjes; Rizzi, Valerio; Parrinello, Michele
      Nature Communications (2022), 13 (1), 5438CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Abstr.: The process of ligand-protein unbinding is crucial in biophysics. Water is an essential part of any biol. system and yet, many aspects of its role remain elusive. Here, we simulate with state-of-the-art enhanced sampling techniques the binding of Benzamidine to Trypsin which is a much studied and paradigmatic ligand-protein system. We use machine learning methods to det. efficient collective coordinates for the complex non-local network of water. These coordinates are used to perform On-the-fly Probability Enhanced Sampling simulations, which we adapt to calc. also the ligand residence time. Our results, both static and dynamic, are in good agreement with expts. We find that the presence of a water mol. located at the bottom of the binding pocket allows via a network of hydrogen bonds the ligand to be released into the soln. On a finer scale, even when unbinding is allowed, another water mol. further modulates the exit time.
    55. 55
      Rizzi, V.; Bonati, L.; Ansari, N.; Parrinello, M. The Role of Water in Host-Guest Interaction. Nat. Commun. 2021, 12 (1), 93,  DOI: 10.1038/s41467-020-20310-0
      55
      The role of water in host-guest interaction
      Rizzi, Valerio; Bonati, Luigi; Ansari, Narjes; Parrinello, Michele
      Nature Communications (2021), 12 (1), 93CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      One of the main applications of atomistic computer simulations is the calcn. of ligand binding free energies. The accuracy of these calcns. depends on the force field quality and on the thoroughness of configuration sampling. Sampling is an obstacle in simulations due to the frequent appearance of kinetic bottlenecks in the free energy landscape. Very often this difficulty is circumvented by enhanced sampling techniques. Typically, these techniques depend on the introduction of appropriate collective variables that are meant to capture the system's degrees of freedom. In ligand binding, water has long been known to play a key role, but its complex behavior has proven difficult to fully capture. In this paper we combine machine learning with phys. intuition to build a non-local and highly efficient water-describing collective variable. We use it to study a set of host-guest systems from the SAMPL5 challenge. We obtain highly accurate binding free energies and good agreement with expts. The role of water during the binding process is then analyzed in some detail.
    56. 56
      Tribello, G. A.; Bonomi, M.; Branduardi, D.; Camilloni, C.; Bussi, G. PLUMED 2: New Feathers for an Old Bird. Comput. Phys. Commun. 2014, 185 (2), 604– 613,  DOI: 10.1016/j.cpc.2013.09.018
      56
      PLUMED 2: New feathers for an old bird
      Tribello, Gareth A.; Bonomi, Massimiliano; Branduardi, Davide; Camilloni, Carlo; Bussi, Giovanni
      Computer Physics Communications (2014), 185 (2), 604-613CODEN: CPHCBZ; ISSN:0010-4655. (Elsevier B.V.)
      Enhancing sampling and analyzing simulations are central issues in mol. simulation. Recently, we introduced PLUMED, an open-source plug-in that provides some of the most popular mol. dynamics (MD) codes with implementations of a variety of different enhanced sampling algorithms and collective variables (CVs). The rapid changes in this field, in particular new directions in enhanced sampling and dimensionality redn. together with new hardware, require a code that is more flexible and more efficient. We therefore present PLUMED 2 here-a complete rewrite of the code in an object-oriented programming language (C++). This new version introduces greater flexibility and greater modularity, which both extends its core capabilities and makes it far easier to add new methods and CVs. It also has a simpler interface with the MD engines and provides a single software library contg. both tools and core facilities. Ultimately, the new code better serves the ever-growing community of users and contributors in coping with the new challenges arising in the field.
    57. 57
      Michaud-Agrawal, N.; Denning, E. J.; Woolf, T. B.; Beckstein, O. MDAnalysis: A Toolkit for the Analysis of Molecular Dynamics Simulations. J. Comput. Chem. 2011, 32 (10), 2319– 2327,  DOI: 10.1002/jcc.21787
      57
      MDAnalysis: A toolkit for the analysis of molecular dynamics simulations
      Michaud-Agrawal, Naveen; Denning, Elizabeth J.; Woolf, Thomas B.; Beckstein, Oliver
      Journal of Computational Chemistry (2011), 32 (10), 2319-2327CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      MDAnal. is an object-oriented library for structural and temporal anal. of mol. dynamics (MD) simulation trajectories and individual protein structures. It is written in the Python language with some performance-crit. code in C. It uses the powerful NumPy package to expose trajectory data as fast and efficient NumPy arrays. It has been tested on systems of millions of particles. Many common file formats of simulation packages including CHARMM, Gromacs, Amber, and NAMD and the Protein Data Bank format can be read and written. Atoms can be selected with a syntax similar to CHARMM's powerful selection commands. MDAnal. enables both novice and experienced programmers to rapidly write their own anal. tools and access data stored in trajectories in an easily accessible manner that facilitates interactive explorative anal. MDAnal. has been tested on and works for most Unix-based platforms such as Linux and Mac OS X. It is freely available under the GNU General Public License from http://mdanal.googlecode.com. © 2011 Wiley Periodicals, Inc. J Comput Chem 2011.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c10258.

    • Alternating access mechanisms, collective variable design, and relevant TM helices sequence alignment, and further analysis of the equilibrium and enhanced sampling MD simulations (state stability, salt-bridge formation and water content, and others) (PDF)

    • Angular motion for the rocking-bundle mechanism (MP4)

    • Presence of water-permeable states (MP4)


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