Quantifying Permeation of Small Charged Molecules across Channels: Electrophysiology in Small Volumes
- Jiajun WangJiajun WangDepartment of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, GermanyMore by Jiajun Wang
- ,
- Lorraine BenierLorraine BenierDepartment of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, GermanyMore by Lorraine Benier
- , and
- Mathias Winterhalter*Mathias Winterhalter*E-mail: [email protected]Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, GermanyMore by Mathias Winterhalter
Copyright © 2022 American Chemical Society. This publication is licensed under CC-BY-NC-ND.
This publication is Open Access under the license indicated. Learn More
Abstract
A major bottleneck in the development of small-molecule antibiotics is to achieve good permeability across the outer membrane in Gram-negative bacteria. Optimization with respect to permeability surprisingly lacks appropriate methods. Recently, we proposed to use the diffusion potential for charged molecules created by their difference in electrophoretic mobility while crossing the outer membrane channel under a concentration gradient. The latter provides semiquantitative values, but the current available setups require large volumes and thus exclude several classes of molecules. Here we propose a simple approach of capturing proteoliposomes at the aperture of glass surface (planar aperture or conical glass capillary) by decreasing the necessary volume below 50 μL. We measured the transport of two charged molecules sulbactam and ceftazidime across the two major porins in Escherichia coli. Both molecules were observed to permeate through these porins, with sulbactam having a higher permeability.
Introduction
ARTICLE SECTIONS
Antimicrobial-resistant bacteria are serious threats to public health and entered the agenda of many organizations like WHO and others. To reach the target of antimicrobial agents, bacteria cell walls are the first barrier to be penetrated. In particular, Gram-negative species (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (1)) with their double-wall structure often provide an unsurmountable barrier to small molecules. As shown previously, the outer-membrane proteins such as OmpF and OmpC from E. coli facilitate the passive entry of small antibiotic molecules. Both porins are composed of three identical water-filled β-barrel monomers. The structure of OmpF revealed by Rosenbusch and co-worker (2,3) revealed that the monomer is composed of 16 antiparallel β-barrels that span in the membrane. There are eight long loops connecting each pair of β-sheet from the extracellular side. Typically, the loop 3 (L3) folds back into the channel lumen and creates a 7–11 Å construction region, which is the narrowest part in the hollow. This loop is mainly composed of charged amino acid groups that make the channel slightly cation selective in potassium chloride solution. (4) OmpC, an ortholog of OmpF, shares 60% identical sequence to OmpF. (5) The crystal structure indicates a smaller pore size and a larger cation ion selectivity (3,4) which corresponds to its high osmotic expression condition. (6)
Mass spectrometry has recently seen a huge progress in the quantification of small amount of substrates accumulated inside the cells, (7) but with limited information on particular porin permeability. To characterize the permeation of small hydrophilic molecules through a particular channel, the so-called liposome swelling assay (5) with a fluorescence readout is often used. This requires large amounts of substances (10 mM) and have requirements on molecule charge states. Following the previous work performed in our laboratory, (8−10) here we optimize the method using electrophysiology patch clamp to investigate the permeability flux through the porins that reconstitute liposomes. To quantify the flux of charged compounds through porins, one needs to first generate a concentration gradient across the membrane and measure the transmembrane potential. The membrane potential caused by the unbalanced flux of cations versus anions is due to different ion mobility inside the porins. (8,9) A simplified theory treating the flux of ions under external voltage and a concentration gradient gives the relative flux densities and thus allows quantitative conclusions on the permeation. Recording the reversal potential is experimentally easy. Assuming a homogeneous electric field in the channel and no interaction among the ions or with the channel wall allows to use the Goldman−Hodgkin−Katz (GHK) equation (11) to describe the ion current under an external electric field and a fixed concentration gradient. Typically, a bi-ionic or a tri-ionic system is applied to simplify the system. In general, a concentration difference of millimolar is the minimum requirement to raise the transmembrane potential to millivolt being detected. Such an approach is label-free, uses only simple theoretical assumption, and is easily accessible. However, standard equipment requires at least a concentration gradient of a few millimolar to accurately measure the membrane potentials within a few millivolt. To date, most of the available instruments require large volume usage and, obviously, this excludes many classes of molecules in terms of the quantity of drug consumption. We hereby seek other approaches using the same concept but miniaturizing the platform to reduce the consumption of the total amount of substrates. In the future, such a design will allow upscaling using a microfluidic array, (12,13) leading to higher reproducibility and success rate for our broad-spectrum antibiotic screening purposes.
Results and Discussion
ARTICLE SECTIONS
Planar Lipid Bilayer Formation Using Port-a-Patch
The commercially available bilayer setup, the Port-a-patch (Nanion Technologies GmbH, Munich, Germany), requires less than 50 μL solution to make the experiment successful. It is achieved by fusing a giant unilamellar vesicle (GUV) to the aperture of a horizontal planar borosilicate glass chip. To avoid different electrode polarization due to the different ion concentration on each side, we added a salt bridge to eliminate the artifacts from the measuring tools. To achieve this, we coated the electrodes with 1.5% Agarose soaked under measuring condition (Figure 1a).
Following previous studies, (14,15) we used borosilicate glass chip with a fabricated 1 μm spherical opening and mounted on the Port-a-patch. The bottom side of the chip sees the electrode, which is connected to the headstage (trans), and experiences the atmospheric pressure adjusted from a suction unit. The top surface of the chip is the reference electrode (cis). To initiate the experiment, 5 μL GUV solution were added to the cis side together with another 5 μL NaCl buffer and then a negative pressure (−25 mbar) was applied immediately to pull the vesicles toward the glass chip. The seal resistance above 1 GΩ is obtained when a vesicle is successfully fused on the chip forming a bilayer. The cis side was then perfused with a buffer solution several times to eliminate the sorbitol introduced from the GUV solution. The volume can be as low as 30–50 μL to achieve the goal of low substrate consumption.
Proteoliposomes-containing membrane proteins were introduced to the electrical ground (cis, or upper half cell on the schema) side of the system. In previous studies, we have shown the orientation of the reconstituted protein and channel activity on the Port-a-patch (i.e., the extracellular side of the protein is the same side as the proteoliposome addition). (15,16) Such an observation is similar to the protein reconstituted into the traditional planar lipid bilayer. (17) A stable baseline of the recording is an indicator of neither the current leakage from the sealing nor the current depletion from the electrodes. The symmetric concentration of 100 mM NaCl pH 6.0 was measured as control experiments (Figure 1b).
Whole GUV Patch Clamp
Another approach is to patch GUVs with a micropipette. The micropipette with a 1 μm diameter opening was fabricated by laser pulling of borosilicate glass capillaries (OD = 1.0 mm, ID = 0.58 mm, length = 10 cm, Sutter Instrument Co., Novato, CA) using a P-2000 capillary puller (Sutter Instrument Co., Novato, CA). A two-step process was used for microcapillary fabrication (see the Supporting Information for more detail). The scanning electron microscopy (SEM) characterization was performed by using the Zeiss Ultra Plus scanning electron microscope (Carl Zeiss, Oberkochen, Germany). The pipettes were sputtered with Au for 20 s before SEM characterizations (Figure 2).
Figure 1
Figure 1. Schematic diagram of the chip configuration on the Port-a-patch. (A) An agarose salt bridge has been used together with the ground electrode. A salt bridge filled with 1.5% agarose is used to cover the ground electrode to eliminate the polarization. (B) A typical GUV has been captured on a borosilicate chip forming the bilayer.
Figure 2
Figure 2. SEM characterization of the microcapillary pulled tip. The pipettes were sputtered with Au for 20 s before SEM characterizations (figure kindly provided by Long’s group, East China University of Science and Technology, Shanghai, China).
Previously GUVs with reconstituted OmpF channels were patched using glass capillaries. Single channel activity recording was achieved by optimizing the concentration of the channel protein during GUV incubation. (18) Unilamellar vesicles are considered because of their bilayer shell and confined small intracellular volume and a relatively controllable geometry. The GUV patch clamping has been achieved by mounting the pulled borosilicate micropipette to the headstage controlled by a micromanipulator (PCS 6200, Burleigh Instrument, Fishers, NY). The capillary was filled with an intercellular solution and attached to the Axon headstage connected to an Ag/AgCl electrode. (18,19) Electro-osmotic shock can cause GUV rupture, (20,21) and the lipid diffuse inside the capillary due to the lack of a membrane skeleton (Figure 3B,C). Bovine serum albumin (BSA) pretreatment is necessary to prevent lipid diffusion in the capillary (19) (i.e., GUVs formed in presence of 2 mg/mL BSA and, in addition, coating the interior of the pipette with BSA solution). As an example, we successfully patched a 20 μm GUV using our homemade device (Figure S2). Before patching a GUV, a minor positive pressure (+5 mbar) was applied to keep the interior clean. The headstage was controlled via the micromanipulator, which allowed the manipulation of the vesicle under a microscope. A negative pressure (from −5 to −15 mbar) was then applied to move the vesicle toward the tip of the micropipette. At this moment, the value of resistance measured by the PClamp (Axon Instruments, Forest City, CA) gradually exceeded 2 GΩ. The vesicle should remain stable at the capillary tip for at least 1 min without shrinking or rupturing before proceeding with the experiments.
Figure 3
Figure 3. (A) Schematic diagram of the whole GUV patch clamp. (B) A vesicle got captured at the microcapillary tip. (C) An unbalanced osmotic pressure causes the vesicle to shrink 240 s after patching, reducing the size of the vesicle to less than 5 μm. The seal resistance exceeded gigaohm gradually. (D) BSA treatment: A vesicle formed in a BSA solution is patched with a BSA coated microcapillary shows no obvious shrinkage (see Whole GUV Patch Clamp for details).
After a vesicle is successfully patched by the micropipette in the whole-cell mode, the pressure was decreased to gain the seal resistance above one gigaohm. As illustrated in the Supporting Information, the capacitance of the DPhPC vesicles is stable at 0.42 ± 0.05 μF/cm2. A 25% lower capacitance compared to the DPhPC bilayer capacitance (0.56 μF/cm2) (22) is attributed to the system capacitance introduced additionally from capillary, solvent, and electronic components. The vesicle eventually ruptured as soon as the pressure decreased to lower than −30 mbar.
Quantifying the Permeation of Small Molecules across Channel
To elucidate the reproducibility of different techniques, we first measured the ion selectivity of both OmpF and OmpC. Under bi-ionic (i.e., Na+, Cl–) condition, using asymmetric concentrations 
, the reversal potential is obtained directly from the I–V plot (Figure S3). The reversal potential of OmpF and OmpC at such an asymmetric condition read 5.9 and 7.0 mV, respectively, using Port-a-patch, whereas 6.2 and 7.2 mV, respectively, using the patch clamp technique.
, the reversal potential is obtained directly from the I–V plot (Figure S3). The reversal potential of OmpF and OmpC at such an asymmetric condition read 5.9 and 7.0 mV, respectively, using Port-a-patch, whereas 6.2 and 7.2 mV, respectively, using the patch clamp technique.Introducing the experimental parameter into eq 2 gives the relative permeability through OmpF 
and 
. However, for OmpC, the permeability are 
and 
and in agreement with the published results. (8,9,23)
and . However, for OmpC, the permeability are and and in agreement with the published results. (8,9,23)In a second set of experiment, we recorded ceftazidime, an antibiotic, and sulbactam, a β-lactamase inhibitor (Figure 4). Both molecules are negatively charged in our condition with Na+ as a counterion. After the determination of the relative permeation rate of sodium and chloride through both OmpF and OmpC under bi-ionic conditions, the permeation rate of the substrates becomes accessible to be calculated under tri-ionic condition.
Figure 4
Figure 4. Molecular structure of (A) sulbactam (MW = 233) and (B) ceftazidime (MW = 546).
To settle the tri-ionic condition, 50 mM substrates were dissolved in 100 mM NaCl to yield a total of 150 mM sodium (+1e) counter with 50 mM substrate (−1e) and 100 mM chloride (−1e) at the cis side. Along with the 100 mM NaCl at the trans side of the bilayer, the asymmetric condition generates a transmembrane potential corresponding to permeability. Moving ahead to the two compounds, the transmembrane voltage generated by 50 mM sulbactam (sodium salt) through OmpF using Port-a-patch and GUV patch clamp is 4.5 and 5.0 mV, respectively, whereas that generated through OmpC is 1.0 and 2.0 mV, respectively. The 50 mM ceftazidime (sodium salt) yielded transmembrane voltage of 5.2 and 5.0 mV using Port-a-patch and GUV patch clamp approach, respectively. We again obtained lower transmembrane voltage of 3.5 and 3.3 mV through the OmpC channel. The same chloride concentration on both sides of bilayer makes the net flux negligible. The permeability of sodium and the tested substrates was normalized to chloride, ex ante. According to the determination of sodium permeation rate in previous section, the relative permeability of substrates were then calculated via eq 3 and summarized in Table 1.
Table 1. Zero-Current Transmembrane Voltage Measurements of Both Porins from E. coli Using Different Techniquesa
| techniques | ||||||||
|---|---|---|---|---|---|---|---|---|
| black lipid membrane (BLM)d | Port-a-patch | GUV patch | ||||||
| porins | conductance (nS)b | asymmetric condition ([cis]–[trans])c | transmembrane potential (mV) | p+/p– | transmembrane potential (mV) | p+/p–/psub | transmembrane potential (mV) | p+/p–/psub |
| OmpF | 0.4 | 50 mM NaCl | 5.5 ± 1.2 | 3.3:1 | 5.9 ± 2.0 | 3.7:1 | 6.2 ± 1.7 | 4:1 |
| 50 mM sulbactam-Na | 4.5 ± 1.2 | 3.7:1:1.5 | 5.0 ± 1.2 | 4:1:1.5 | ||||
| 50 mM ceftazidime-Na | 5.2 ± 3.0 | 3.7:1:1.2 | 5.0 ± 2.7 | 4:1:1.5 | ||||
| OmpC | 0.3 | 50 mM NaCl | 7.0 ± 1.5 | 5.2:1 | 7.0 ± 2.5 | 5.3:1 | 7.2 ± 2.0 | 5.6:1 |
| 50 mM sulbactam-Na | 1.0 ± 2.0 | 5.3:1:4.6 | 2.0 ± 1.0 | 5.6:1:4.1 | ||||
| 50 mM ceftazidime-Na | 3.5 ± 1.2 | 5.3:1:2.9 | 3.3 ± 0.9 | 5.6:1:3.2 | ||||
a
The NaCl concentrations were buffered with 20 mM MES and pH of 6.0 was measured at room temperature. BLM technique is measured as a control. All measurements were repeated at least thrice.
b
Electrical conductance measured for single channel under 100 mM NaCl, pH 6.0.
c
Asymmetric condition generated from 100 mM NaCl, pH 6.0. Additional 50 mM analysts are added from the cis (ground) side.
d
BLM was not used to measure valuable substrates due to the large volume assumption.
The permeability of cation (i.e., sulbactam and ceftazidime) obtained from both Port-a-patch and GUV patch clamp methods is similar to that of counterion (i.e., Cl–), in agreement with previous studies. A slight variation does not change the conclusion that the permeability of sulbactam is generally lower than that of sodium ions. Moreover, sulbactam permeates faster through both porins compared to ceftazidime. Interestingly, although OmpC is somewhat smaller and more cation selective, both negatively charged ions show higher rates compared to OmpF, although the net substrate conductivity might vary. This observation suggests that in both cases, the interaction with the channel surface is less pronounced and other effects like electro-osmosis may have a stronger impact. (24,25)
Conclusions
ARTICLE SECTIONS
To quantify the translocation of charged small molecules across the membrane channel, we measured the zero-current transmembrane potential. As this method is based on concentration gradients, reduction of volume to the value below the required quantity (below mg) is a prerequisite to test certain class of molecules, particularly for screening purposes. Using the simple concept of reversal potential measurement, we have characterized the relative permeability ratio of small antimicrobial molecules, sulbactam, and ceftazidime. Both negatively charged molecules have a surprisingly fast diffusion. Sulbactam has a generally stronger permeability through channels compared to ceftazidime mainly due to its small molecular structure. Both molecules have a preference to OmpC over OmpF, although former is more cation selective than the latter. Further details of the molecule–protein interaction could be investigated via in situ analysis on a microscopic time scale. In all, this method has great potential for fast screening of small charged molecules through membrane channels compared to other in vitro electrochemical characterizations.
Last but not least, one of the drawbacks of such a method is the substrate consumption issue. In this manuscript, we also optimized our homemade devices with respect to volume. We show that for the commercially available Port-a-patch, the volume can be decreased to 20–50 and 150 μL using whole GUV patch clamping with a homemade device. The volume consumption in the GUV patch clamping is dominated by the O-ring, which accumulates the buffer.
In the future the required volume could be reduced using multiple pipettes sharing one reservoir. The parallelization of the vesicle patch clamp could be achieved by using several pipettes in parallel or implementing into a microfluidic device to reach the low volumetric consumption. (26) The pros and cons are summarized in Table 2.
Table 2. Comparison of Different Techniques
| technique | |||
|---|---|---|---|
| features | BLM | Port-a-patch | GUV patch clamp |
| minimum volume (cis side) | 2500 μL | 40 μL | 150 μL |
| accessibility (trans side) | yes | yes (addition device) | hardly |
| parallelization | no | yes (14,27) (microfluidic approach) | complex (26) (multiple patch tips) |
| consumables | reusable Teflon thin film | disposable microfluidic chamber | disposable borosilicate glass capillary |
Methods
ARTICLE SECTIONS
Goldman–Hodgkin–Katz Equation Application
To estimate the permeability of charged molecules, we analyze the permeability ratio of the ions. In the simple case of a 1:1 electrolyte (e.g., NaCl), this can be obtained by applying a concentration gradient and recording the voltage needed to zero net ion current. The Goldman–Hodgkin–Katz (GHK) provides a simple relation that allows us to obtain the permeability ratio (11)
By measuring the experimental zero-current transmembrane voltage at the desired asymmetric condition, the relative permeability (Px (x = Na+, Cl–)) ratio between cation (Na+) and anion (Cl–) gives
In case multivalent ions are involved, we rather use the GHK equation for the ion current I and solve the permeability ratio numerically. The ion current I is then given by the sum of all the individual ion currents Ix
where V is the transmembrane voltage, Px is the permeability for the ion x, z is the valence, F (Faraday constant) = 9.6 × 104 mol–1, and R (gas constant) = 8.3 J/(mol K). The experimental input cx,cis and cx,trans are the ion concentrations on the two sides of the membrane.
(1)
(2)
(3)
Proteoliposome Preparation
The whole process of electroformation was controlled by the Vesicle Prep Pro (Nanion Technology GmbH, Munich, Germany) with programmable parameters (i.e., amplitude, frequency, main time, rise time, and fall time). We used indium tin oxide (ITO) slides as electrodes for the GUV electroformation. Ten microliters of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (5 mg/mL) dissolved in chloroform was deposited and spread carefully on the conductive part of the ITO slide. After complete evaporation of chloroform, an O-ring (1 mm thickness) was placed around the dried lipid residue. One molar sorbitol was added to fill the O-ring space. Here, we added 275 μL in accordance with the O-ring size. Another pair of the ITO slide was then covered above the O-ring, with the conductive sides facing each other. During the electroformation period, an AC current (Vpp = 3 V, f = 5 Hz) is applied through the ITO slides and, after 2 h of formation, both the voltage and frequency are gradually decreased to zero within 3 min. Finally, the ITO slide was uncovered and the GUV solution was collected in Eppendorf tubes.
Purified membrane protein either OmpF or OmpC (1.5 mg/mL), as previously described, (28) see also supplement for details, was added directly to the GUV solution. The protein was diluted in 1% n-octyl-oligo-oxyethylene (Octyl-POE) and then added to the GUV solution for incubation. A final concentration of 10–13 mg/mL as described was reached to obtain few channel activities per vesicle fusion as described previously. (15,18,29) The protein–GUV mixture was gently vortexed and placed at room temperature for 2–4 h and then 500 mg/mL biobeads (Bio-Rad Laboratories, Inc., Hercules, CA) was added to the solution and further incubated at 4 °C overnight for the removal of excess detergent. Finally, after brief centrifugation, the proteoliposome-rich supernatant was collected.
Supporting Information
ARTICLE SECTIONS
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01611.
Patched vesicles electrical behavior and microscopic image; pipette pulling parameters as well as the permeability calculation approaches; purification of membrane proteins (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
ARTICLE SECTIONS
- Jiajun Wang - Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, Germany;
http://orcid.org/0000-0003-3420-6376
Acknowledgments
ARTICLE SECTIONS
J.W. is supported from the Innovative Medicines Initiative Joint Undertaking under Grant Agreement No. 115525, resources which are composed of financial contribution from European Union’s seventh framework programme (FP7/2007-2013), and EFPIA companies. We thank Prof. Yi-Tao Long’s group from East China University of Science and Technology for providing home-designed nanopore fabrication and detection instrumentation. We further acknowledge support from the project “Boomer” - Bacterial periplasmic organelles and outer membrane vesicles (ZF4176703AJ6) by the Federal Ministry for Economic Affairs and Energy (BMWi) under the framework of the Central Innovation Programme for SMEs (ZIM).
References
ARTICLE SECTIONS
This article references 29 other publications.
- 1Mortimer, P. G.; Piddok, L. J. The accumulation of five antibacterial agents in porin-deficient mutants of Escherichia coli. J. Antimicrob. Chemother. 1993, 32, 195– 213, DOI: 10.1093/jac/32.2.195[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXms1Khurc%253D&md5=72a47ea868a250c0f3a59a16bdf778b7The accumulation of five antibacterial agents in porin-deficient mutants of Escherichia coliMortimer, Peter G. S.; Piddock, Laura J. V.Journal of Antimicrobial Chemotherapy (1993), 32 (2), 195-213CODEN: JACHDX; ISSN:0305-7453.A library of isogenic mutants contg. Mud9-induced deletions of the structural and regulatory genes for the porin proteins OmpF and OmpC of E. coli was constructed. The accumulation of norfloxacin, tetracycline, chloramphenicol, cephalothin, and cefoxitin was measured with each strain and shown to be reproducible with low exptl. std. deviations, such that the roles of OmpF, OmpC, and PhoE in the accumulation of these agents were detd. The loss of OmpF reduced accumulation of norfloxacin, tetracycline, cephalothin, and cefoxitin by 16-60% compared to the wild-type parent strain, but reduced accumulation of chloramphenicol by <10%. The loss of OmpC reduced accumulation of cephalothin and cefoxitin by 13 and 34%, resp., compared to the wild-type parent strain, but had little effect on the accumulation of norfloxacin, chloramphenicol, and tetracycline (<3%). The loss of both OmpF and OmpC (ompR) reduced accumulation of norfloxacin, chloramphenicol, tetracycline, cephalothin, and cefoxitin by 36-68%. However, the presence of PhoE in the absence of both OmpF and OmpC enhanced accumulation of these 5 agents to 52-119% of the concns. accumulated by the wild-type strain. These data suggest that OmpF is the preferred route of entry for 3 of the antibiotics studied, but not for chloramphenicol and tetracycline, which utilize both porins equally well. The high levels of accumulation (30-64%) of all 5 antibiotics in the absence of all major porins suggest that an alternative mechanism(s) of accumulation is available.
- 2Cowan, S. W.; Garavito, R.; Jansonius, J.; Jenkins, J.; Karlsson, R.; König, N.; Pai, E.; Pauptit, R.; Rizkallah, P.; Rosenbusch, J. The structure of OmpF porin in a tetragonal crystal form. Structure 1995, 3, 1041– 1050, DOI: 10.1016/S0969-2126(01)00240-4[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptVagtr8%253D&md5=5bd39aec8d5ef8c06336cb0b617950b6The structure of OmpF porin in a tetragonal crystal formCowan, S. W.; Garavito, R. M.; Jansonius, J. N.; Jenkins, J. A.; Karlsson, R.; Koenig, N.; Pai, E. F.; Pauptit, R. A.; Rizkallah, P. J.; et al.Structure (London) (1995), 3 (10), 1041-50CODEN: STRUE6; ISSN:0969-2126. (Current Biology)OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic mols., such as nutrients and waste products, across the outer membrane of Escherichia coli. Very few membrane proteins have been crystd. in three dimensions, yet this stable protein can be obtained in several crystal forms. Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows the authors to explore the integrity of the structure outside the natural membrane environment. The structure of OmpF porin in a tetragonal crystal form with two trimers per asym. unit has been detd. at 3.2 Å resoln. and compared with that obtained previously in a trigonal crystal form. The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice. The trimer structure is virtually identical in both. This comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints, and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided that starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.
- 3Cowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R.; Jansonius, J.; Rosenbusch, J. Crystal structures explain functional properties of two E. coli porins. Nature 1992, 358, 727– 733, DOI: 10.1038/358727a0[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XlsVGltbo%253D&md5=64f9b453e89092fb7edb57fc07bb2332Crystal structures explain functional properties of two E. coli porinsCowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R. A.; Jansonius, J. N.; Rosenbusch, J. P.Nature (London, United Kingdom) (1992), 358 (6389), 727-33CODEN: NATUAS; ISSN:0028-0836.Porins form aq. channels that aid the diffusion of small hydrophilic mols. across the outer membrane of Gram-neg. bacteria. The crystal structures of matrix porin and phosphoporin both reveal trimers of identical subunits, each subunit consisting of a 16-stranded anti-parallel β-barrel contg. a pore. A long loop inside the barrel contributes to a constriction of the channel where the charge distribution affects ion selectivity. The structures explain at the mol. level functional characteristics and their alterations by known mutations.
- 4Benz, R.; Schmid, A.; Hancock, R. Ion selectivity of gram-negative bacterial porins. J. Bacteriol. 1985, 162, 722– 727[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXitFKquro%253D&md5=545bea1d2bc6247febcba25ab9f07e90Ion selectivity of gram-negative bacterial porinsBenz, Roland; Schmid, Angela; Hancock, Robert E. W.Journal of Bacteriology (1985), 162 (2), 722-7CODEN: JOBAAY; ISSN:0021-9193.Twelve different porins from the gram-neg. bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single channel conductance between 1.5 and 6 nS in 1M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free soln. Thus, the single-channel conductances of the individual porins could be used to est. the effective channel diams. of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes contg. individual porins gave results that were consistent with the conclusions drawn from the single-channel expts. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aq. phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examd., including all of the known constitutive porins of the 4 gram-neg. bacteria studied, were cation selective with a 3-40-fold preference for K+ over Cl-.
- 5Nikaido, H. Molecular Basis of Bacterial Outer Membrane Permeability Revisited. Microbiol. Mol. Biol. Rev. 2003, 67, 593– 656, DOI: 10.1128/MMBR.67.4.593-656.2003[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmt1Sqsg%253D%253D&md5=6b2e709a18bc697e20070aafb412b06fMolecular basis of bacterial outer membrane permeability revisitedNikaido, HiroshiMicrobiology and Molecular Biology Reviews (2003), 67 (4), 593-656CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)A review. Gram-neg. bacteria characteristically are surrounded by an addnl. membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compds. and at the same time to allow the influx of nutrient mols. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in mol. detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide phospholipid asym. bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compds., owing to our increasing knowledge about the chem. of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.
- 6Liu, X.; Ferenci, T. An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation. Microbiology 2001, 147, 2981– 2989, DOI: 10.1099/00221287-147-11-2981
- 7Philipsen, M. H.; Phan, N. T.; Fletcher, J. S.; Malmberg, P.; Ewing, A. G. Mass Spectrometry Imaging Shows Cocaine and Methylphenidate have Opposite Effects on Major Lipids in Drosophila Brain. ACS Chem. Neurosci. 2018, 1462– 1468, DOI: 10.1021/acschemneuro.8b00046[ACS Full Text
], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvVKrtLc%253D&md5=adfbcf34552d90d0947438ae6e1ada85Mass Spectrometry Imaging Shows Cocaine and Methylphenidate Have Opposite Effects on Major Lipids in Drosophila BrainPhilipsen, Mai H.; Phan, Nhu T. N.; Fletcher, John S.; Malmberg, Per; Ewing, Andrew G.ACS Chemical Neuroscience (2018), 9 (6), 1462-1468CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to study the effects of cocaine vs. methylphenidate administration on both the localization and abundance of lipids in Drosophila melanogaster brain. A J105 ToF-SIMS with a 40 keV gas cluster primary ion source enabled the authors to probe mol. ions of biomols. on the fly with a spatial resoln. of ∼3 μm, giving the authors unique insights into the effect of these drugs on mol. lipids in the nervous system. Significant changes in phospholipid compn. were obsd. in the central brain for both. Principal components image anal. revealed that changes occurred mainly for phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols. When the lipid changes caused by cocaine were compared with those induced by methylphenidate, it was shown that these drugs exert opposite effects on the brain lipid structure. The authors speculate that this might relate to the mol. mechanism of cognition and memory. - 8Ghai, I.; Pira, A.; Scorciapino, M. A.; Bodrenko, I.; Benier, L.; Ceccarelli, M.; Winterhalter, M.; Wagner, R. General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpF. J. Phys. Chem. Lett. 2017, 8, 1295– 1301, DOI: 10.1021/acs.jpclett.7b00062[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVCmtbo%253D&md5=1cb3e491a3bb31fa660a8aae0d2891c1General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpFGhai, Ishan; Pira, Alessandro; Scorciapino, Mariano Andrea; Bodrenko, Igor; Benier, Lorraine; Ceccarelli, Matteo; Winterhalter, Mathias; Wagner, RichardJournal of Physical Chemistry Letters (2017), 8 (6), 1295-1301CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)A major challenge in the discovery of the new antibiotics against Gram-neg. bacteria is to achieve sufficiently fast permeation to avoid high doses causing toxic side effects. So far, suitable assays for quantifying the uptake of charged antibiotics into bacteria are lacking. The authors apply electrophysiol. zero-current assay using concn. gradients of β-lactamase inhibitors combined with single channel conductance to quantify their flux rates through OmpF. Mol. dynamic simulations provide in addn. details on the interactions between the nanopore wall and the charged solutes. In particular, the interaction barrier for three β-lactamase inhibitors is surprisingly as low as 3-5 kcal/mol, and only slightly above the diffusion barrier of ions such as chloride. Within the authors' macroscopic const. field model, the authors det. that at zero-membrane potential a concn. gradient of 10 μM of avibactam, sulbactam or tazobactam can create a flux rates of roughly 620 mols./s per OmpF trimer.
- 9Ghai, I.; Winterhalter, M.; Wagner, R. Probing transport of charged β-lactamase inhibitors through OmpC, a membrane channel from E. coli. Biochem. Biophys. Res. Commun. 2017, 484, 51– 55, DOI: 10.1016/j.bbrc.2017.01.076[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVKisrw%253D&md5=d1a6d0bf3d9158faca51ab394fe55049Probing transport of charged lactamase inhibitors through OmpC, a membrane channel from E. coliGhai, Ishan; Winterhalter, Mathias; Wagner, RichardBiochemical and Biophysical Research Communications (2017), 484 (1), 51-55CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)One of the major causes of antibiotic resistance in the Gram-neg. bacteria is the low permeability across the outer membrane. Currently a main bottleneck in the development of effective antibiotics is the lack of a general method to quantify permeation which would allow screening for optimal scaffolds. Here, we present a permeation assay based on conventional electrophysiol. The method mainly involves application of concn. gradients of charged mols. with different electrophoretic mobilities through a membrane channel. Thus the unbalanced flux creates an electrostatic potential which provides direct information on relative ion fluxes. The exptl. approach applied here involves measuring zero-current-potentials and the corresponding single channel conductance. For OmpC and the lactamase inhibitor avibactam at a 10 microm gradient the calcd. flux rate at Vm = 0mV was about n = 200 mols./s per OmpC single pore.
- 10Soundararajan, G.; Bhamidimarri, S. P.; Winterhalter, M. Understanding carbapenem translocation through OccD3 (OpdP) of Pseudomonas aeruginosa. ACS Chem. Biol. 2017, 12, 1656– 1664, DOI: 10.1021/acschembio.6b01150[ACS Full Text], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmsVaht7o%253D&md5=19c5c5cf63ff6e87db3bb7dae7c694d6Understanding Carbapenem Translocation through OccD3 (OpdP) of Pseudomonas aeruginosaSoundararajan, Gowrishankar; Bhamidimarri, Satya Prathyusha; Winterhalter, MathiasACS Chemical Biology (2017), 12 (6), 1656-1664CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Pseudomonas aeruginosa utilizes a plethora of substrate specific channels for the uptake of small nutrients. OccD3 (OpdP or PA4501) is an OprD like arginine uptake channel of P. aeruginosa whose role has been implicated in carbapenem uptake. To understand the mechanism of selective permeation we reconstituted single OccD3 channels in planar lipid bilayer and characterized the interaction with Imipenem and Meropenem analyzing the ion current fluctuation in presence of substrates. We performed point mutations in the constriction region of OccD3 to understand the binding and translocation of antibiotic in OccD3. By mutating 2 key residues in the substrate binding sites of OccD3 (located in the internal loop L7 and basic ladder) we emphasize the importance of these residues. We show that carbapenem antibiotics follow a similar path as arginine through the constriction zone and the basic ladder to translocate across OccD3.
- 11Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates Inc, 2001.Google ScholarThere is no corresponding record for this reference.
- 12Braeken, D.; Jans, D.; Huys, R.; Stassen, A.; Collaert, N.; Hoffman, L.; Eberle, W.; Peumans, P.; Callewaert, G. Open-cell recording of action potentials using active electrode arrays. Lab Chip 2012, 12, 4397– 4402, DOI: 10.1039/c2lc40656j[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVCqu73F&md5=a04797d31e56bd089ba5c1516bc598f3Open-cell recording of action potentials using active electrode arraysBraeken, Dries; Jans, Danny; Huys, Roeland; Stassen, Andim; Collaert, Nadine; Hoffman, Luis; Eberle, Wolfgang; Peumans, Peter; Callewaert, GeertLab on a Chip (2012), 12 (21), 4397-4402CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)The investigation of complex communication in cellular networks requires superior measurement tools than those available to date. Electrode arrays integrated onto silicon electronics are increasingly used to measure the elec. activity of cells in an automated and highly parallelized fashion, but they are restricted to recording extracellular potentials. Here, we report on an array of TiN electrodes built using std. silicon electronics for intracellular action potential recording. Intracellular access, possible at each of the 16 384 electrodes on the chip, was accomplished by local membrane electroporation using elec. stimulation with subcellular, micrometer-sized electrodes. Access to the cell interior was transient and could be tuned in duration by adapting the electroporation protocol. Intracellular sensing was found to be minimally invasive in the short and long-term, allowing consecutive intracellular recordings from the same cell over the course of days. Finally, we applied this method to investigate the effect of an ion channel blocker on cardiac elec. activity. This technique opens the door to massively parallel, long-term intracellular recording for fundamental electrophysiol. and drug screening.
- 13Abbott, J.; Ye, T.; Ham, D.; Park, H. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology. Acc. Chem. Res. 2018, 51, 600– 608, DOI: 10.1021/acs.accounts.7b00519[ACS Full Text], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFOrsrc%253D&md5=d321cbe0a2297c60f074421d91654b67Optimizing Nanoelectrode Arrays for Scalable Intracellular ElectrophysiologyAbbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, HongkunAccounts of Chemical Research (2018), 51 (3), 600-608CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Electrode technol. for electrophysiol. has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold std. in studying the fundamental cellular processes underlying the elec. activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large no. of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiol. screening from basic neurobiol. labs. to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the std. top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive no. of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiol., and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful ref. Summing up from the recent studies, the authors will first elucidate the morphol. and assocd. elec. properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which the authors will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chem. modifications, electroporation protocols, and recording/stimulation electronics, as the authors describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. The authors will discuss this theme using a recent development from the groups as an example, where an array of ∼1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiol.
- 14Farre, C.; Stoelzle, S.; Haarmann, C.; George, M.; Brüggemann, A.; Fertig, N. Automated ion channel screening: patch clamping made easy. Expert Opin. Ther. Targets 2007, 11, 557– 565, DOI: 10.1517/14728222.11.4.557[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjt1Sqsrw%253D&md5=df83373ddd57528146cf70740fa22159Automated ion channel screening: patch clamping made easyFarre, Cecilia; Stoelzle, Sonja; Haarmann, Claudia; George, Michael; Brueggemann, Andrea; Fertig, NielsExpert Opinion on Therapeutic Targets (2007), 11 (4), 557-565CODEN: EOTTAO; ISSN:1472-8222. (Informa Healthcare)A review. Efficient high resoln. techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resoln. but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technol. meets the need of drug developers for higher throughput and facilitates new exptl. approaches in ion channel research. Specifically, Nanion's electrophysiol. workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated exptl. possibilities, such as accurate and fast ligand application, temp. control and internal soln. exchange. This article describes the chip-based patch clamp technol. and its usefulness in ion channel drug screening and academic research.
- 15Kreir, M.; Farre, C.; Beckler, M.; George, M.; Fertig, N. Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes. Lab Chip 2008, 8, 587– 595, DOI: 10.1039/b713982a[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjslyksb0%253D&md5=e8f6cb817f1133132a329c8a2404421dRapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomesKreir, Mohamed; Farre, Cecilia; Beckler, Matthias; George, Michael; Fertig, NielsLab on a Chip (2008), 8 (4), 587-595CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp anal., like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and obsd. the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin contg. lipid bilayers is of potential for the efficient electrophysiol. characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.
- 16Mahendran, K. R.; Kreir, M.; Weingart, H.; Fertig, N.; Winterhalter, M. Permeation of Antibiotics through Escherichia coli OmpF and OmpC Porins Screening for Influx on a Single-Molecule Level. J. Biomol. Screening 2010, 15, 302– 307, DOI: 10.1177/1087057109357791[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFWjs74%253D&md5=6f51572d413a079fdff09182751b8072Permeation of antibiotics through Escherichia coli OmpF and OmpC porins: screening for influx on a single-molecule levelMahendran, Kozhinjampara R.; Kreir, Mohamed; Weingart, Helge; Fertig, Niels; Winterhalter, MathiasJournal of Biomolecular Screening (2010), 15 (3), 302-307CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)A chip-based automated patch-clamp technique provides an attractive biophys. tool to quantify solute permeation through membrane channels. Proteo-giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics-cephalosporins and fluoroquinolones-through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to det. the mode of permeation. The chip-based setup allows rapid soln. exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-mol. level.
- 17Ionescu, S. A.; Lee, S.; Housden, N. G.; Kaminska, R.; Kleanthous, C.; Bayley, H. Orientation of the OmpF Porin in Planar Lipid Bilayers. ChemBioChem 2017, 18, 554– 562, DOI: 10.1002/cbic.201600644
- 18Gornall, J. L.; Mahendran, K. R.; Pambos, O. J.; Steinbock, L. J.; Otto, O.; Chimerel, C.; Winterhalter, M.; Keyser, U. F. Simple reconstitution of protein pores in nano lipid bilayers. Nano Lett. 2011, 11, 3334– 3340, DOI: 10.1021/nl201707d[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptVKrt7g%253D&md5=88a56c5100b6a12a732116076299f6dcSimple Reconstitution of Protein Pores in Nano Lipid BilayersGornall, Joanne L.; Mahendran, Kozhinjampara R.; Pambos, Oliver J.; Steinbock, Lorenz J.; Otto, Oliver; Chimerel, Catalin; Winterhalter, Mathias; Keyser, Ulrich F.Nano Letters (2011), 11 (8), 3334-3340CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors developed a new, simple and robust approach for rapid screening of single mol. interactions with protein channels. The authors' glass nanopipets can be fabricated simply by drawing glass capillaries in a std. pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ∼140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by elec. and mech. forces. The authors demonstrate the functional reconstitution of the Escherichia coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-mol. level, demonstrating the ability of this method to detect single mol. interactions with protein channels. High-resoln. conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-mol. studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.
- 19Garten, M.; Mosgaard, L. D.; Bornschlögl, T.; Dieudonné, S.; Bassereau, P.; Toombes, G. E. Whole-GUV patch-clamping. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 328– 333, DOI: 10.1073/pnas.1609142114[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFentrnK&md5=757fba9d98f5630604eb235709f0de83Whole-GUV patch-clampingGarten, Matthias; Mosgaard, Lars D.; Bornschlogl, Thomas; Dieudonne, Stephane; Bassereau, Patricia; Toombes, Gilman E. S.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (2), 328-333CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiol. high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiol., but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipet and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipet. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipet surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compns. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiol. range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane compn., tension, and shape.
- 20Su, J.; Zhao, Y.; Fang, C.; Shi, Y. Asymmetric osmotic water permeation through a vesicle membrane. J. Chem. Phys. 2017, 146, 204902 DOI: 10.1063/1.4983749[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotlWitLk%253D&md5=772b89cc302acf861ac23f0c08fd7059Asymmetric osmotic water permeation through a vesicle membraneSu, Jiaye; Zhao, Yunzhen; Fang, Chang; Shi, YueJournal of Chemical Physics (2017), 146 (20), 204902/1-204902/7CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Understanding water permeation through a cell membrane is of primary importance for biol. activities and a key step to capture its shape transformation in salt soln. Here, we reveal the dynamical behaviors of osmotically driven transport of water mols. across a vesicle membrane by mol. dynamics simulations. Of particular interest is that the water transport in and out of vesicles is highly distinguishable given the osmotic forces are the same, suggesting an asym. osmotic transportation. This asym. phenomenon exists in a broad range of parameter space such as the salt concn., temp., and vesicle size, and can be ascribed to the similar asym. potential energy of lipid-ion, lipid-water, lipid-soln., lipid-lipid, and the lipid-lipid energy fluctuations. Specifically, the water flux has a linear increase with the salt concn., similar to the prediction by Nernst-Planck equation or Fick's 1st law. Furthermore, due to the Arrhenius relation between the membrane permeability and temp., the water flux also exhibits excellent Arrhenius dependence on the temp. Meanwhile, the water flux shows a linear increase with the vesicle surface area since the flux amt. across a unit membrane area should be a const. Finally, we also present the anonymous diffusion behaviors for the vesicle itself, where transitions from normal diffusion at short times to subdiffusion at long times are identified. These results provide significant new phys. insights for osmotic water permeation through a vesicle membrane and are helpful for future exptl. studies. (c) 2017 American Institute of Physics.
- 21Su, J.; Yao, Z.; de la Cruz, M. O. Vesicle Geometries Enabled by Dynamically Trapped States. ACS Nano 2016, 10, 2287– 2294, DOI: 10.1021/acsnano.5b06991[ACS Full Text], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Khsbc%253D&md5=943c6f3d33295ec34564244abf7dd97cVesicle Geometries Enabled by Dynamically Trapped StatesSu, Jiaye; Yao, Zhenwei; Olvera de la Cruz, MonicaACS Nano (2016), 10 (2), 2287-2294CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The authors design dynamic protocols for enlarging the shape space of both fluid and cryst. vesicles beyond the equil. zone. By removing water from within the vesicle at different rates, the authors numerically produced a series of dynamically trapped stable vesicle shapes for both fluid and cryst. vesicles in a highly controllable fashion. In cryst. vesicles that are continuously dehydrated, simulations show the initial appearance of small flat areas over the surface of the vesicles that ultimately merge to form fewer flat faces. In this way, the vesicles transform from a fullerene-like shape into various faceted polyhedrons. The authors perform anal. elasticity anal. to show that these salient features are attributable to the cryst. nature of the vesicle. The potential to use dynamic protocols, such as those used in this study, to engineer vesicle shape transformations is helpful for exploiting the richness of vesicle geometries for desired applications.
- 22Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques. Biochim. Biophys. Acta, Biomembr. 1996, 1279, 169– 180, DOI: 10.1016/0005-2736(95)00274-X[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhsFOjtbc%253D&md5=f62efc4a6b801c312e9ebef4e6951cbcImpedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniquesSteinem, Claudia; Janshoff, Andreas; Ulrich, Wolf-Peter; Sieber, Manfred; Galla, Hans-JoachimBiochimica et Biophysica Acta, Biomembranes (1996), 1279 (2), 169-80CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)One topic of this study is the comparison of different prepn. techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid soln. in n-decane/isobutanol, (iii) by the lipid/detergent diln. technique or (i.v.) by fusion of vesicles. (2) Charged mols. carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Neg. charged surfaces of 3-mercaptopropionic acid were excellent substrates that allow the attachment of planar lipid bilayers by applying pos. charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or neg. charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If pos. charged first monolayers of mercaptoethylammoniumhydrochloride were used the authors were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles contg. 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A crit. amt. of more than 50 mol% of DMPTE was necessary to form a solid supported lipid bilayer. Bilayers obtained with these different prepn. techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. The authors used a DODAB-bilayer for the reconstitution of gramicidin D channels. By CD measurements the authors verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance anal.
- 23Benz, R.; Schmid, A.; Nakae, T.; Vos-Scheperkeuter, G. Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J. Bacteriol. 1986, 165, 978– 986, DOI: 10.1128/jb.165.3.978-986.1986[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XhsVGhs7s%253D&md5=34eb9b2235e3f4a53426e6aad938bd00Pore formation by LamB of Escherichia coli in lipid bilayer membranesBenz, Roland; Schmid, Angela; Nakae, Taiji; Vos-Scheperkeuter, Greetje H.Journal of Bacteriology (1986), 165 (3), 978-86CODEN: JOBAAY; ISSN:0021-9193.Lipid bilayer expts. were performed in the presence of different E. coli LamB prepns. These LamB prepns. formed 2 types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised ∼1-6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1M KCl. These pores could be completely blocked by the addn. of maltose and maltodextrins. Titrn. of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 × 10-4M for maltotriose. Apparently, the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.
- 24Bhamidimarri, S. P.; Prajapati, J. D.; van den Berg, B.; Kleinekathoefer, U.; Winterhalter, M. Electro-Osmotic Driven Kinetics of Cyclodextrin through the CymA Channel. Biophys. J. 2016, 110, 115a, DOI: 10.1016/j.bpj.2015.11.673
- 25Bhamidimarri, S. P.; Prajapati, J. D.; van den Berg, B.; Winterhalter, M.; Kleinekathöfer, U. Role of electroosmosis in the permeation of neutral molecules: CymA and cyclodextrin as an example. Biophys. J. 2016, 110, 600– 611, DOI: 10.1016/j.bpj.2015.12.027[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnvFGrtQ%253D%253D&md5=4c1ed5c0adb3525daf20b501100163f1Role of Electroosmosis in the Permeation of Neutral Molecules: CymA and Cyclodextrin as an ExampleBhamidimarri, Satya Prathyusha; Prajapati, Jigneshkumar Dahyabhai; van den Berg, Bert; Winterhalter, Mathias; Kleinekathoefer, UlrichBiophysical Journal (2016), 110 (3), 600-611CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)To quantify the flow of small uncharged mols. into and across nanopores, one often uses ion currents. The resp. ion-current fluctuations caused by the presence of the analyte make it possible to draw some conclusions about the direction and magnitude of the analyte flow. However, often this flow appears to be asym. with respect to the applied voltage. As a possible reason for this asymmetry, the authors identified the electroosmotic flow (EOF), which is the water transport assocd. with ions driven by the external transmembrane voltage. As an example, the authors quantify the contribution of the EOF through a nanopore by studying the permeation of α-cyclodextrin through CymA, a cyclodextrin-specific channel from Klebsiella oxytoca. To understand the results from electrophysiol. on a mol. level, all-atom mol. dynamics simulations were used to detail the effect of the EOF on substrate entry to and exit from a CymA channel in which the N-terminus has been deleted. The combined exptl. and computational results strongly suggest that one needs to account for the significant contribution of the EOF when analyzing the penetration of cyclodextrins through the CymA pore. This example study at the same time points to the more general finding that the EOF needs to be considered in translocation studies of neutral mols. and, at least in many cases, should be able to help in discriminating between translocation and binding events.
- 26Guzel, F. D.; Citak, F. Development of an On-Chip Antibiotic Permeability Assay With Single Molecule Detection Capability. IEEE Trans. Nanobiosci. 2018, 17, 155– 160, DOI: 10.1109/TNB.2018.2809592
- 27Saha, S. C.; Powl, A. M.; Wallace, B. A.; de Planque, M. R.; Morgan, H. Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip. Biomicrofluidics 2015, 9, 014103 DOI: 10.1063/1.4905313[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslCnug%253D%253D&md5=54bd1e95233b8967d3dbee7be20e1361Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chipSaha, Shimul C.; Powl, Andrew M.; Wallace, B. A.; de Planque, Maurits R. R.; Morgan, HywelBiomicrofluidics (2015), 9 (1), 014103/1-014103/11CODEN: BIOMGB; ISSN:1932-1058. (American Institute of Physics)We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiol. screening of ion channels and transporters. A passive pumping method is used to flow microliter vols. of ligand soln. across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 μl/min. Phospholipid bilayers are formed across a photolithog. defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with β-cyclodextrin (β-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for detg. IC50 values. The assays are fast (30 min for a complete IC50 curve) and simple and require very small amts. of compds. (100 μg in 15 μl). The technol. can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores. (c) 2015 American Institute of Physics.
- 28Rosenbusch, J. P. Characterization of the major envelope protein from Escherichia coli regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J. Biol. Chem. 1974, 249, 8019– 8029[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2MXmvFCmtw%253D%253D&md5=cd26fa2c682ad0570d0058f2d42a78afCharacterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate bindingRosenbusch, Jurg P.Journal of Biological Chemistry (1974), 249 (24), 8019-29CODEN: JBCHA3; ISSN:0021-9258.The major envelope protein from E. coli was purified by differential heat extn. in dodecyl sulfate and subsequently freed of the detergent. The polypeptide was homogenous and had a mass of 36,500 daltons. Homogeneity was based on 4 criteria, 3 of which were independent of its behavior in detergents. The mass of the protein was accounted for entirely, or nearly entirely, by the mass of its constituent amino acids. Probably, dodecyl sulfate is bound in amts. corresponding to those found in most polypeptides. The protein was also isolated in assocn. with the rigid layer of the cell by extn. of cell envelopes in 2% dodecyl sulfate at 60°. This complex was composed of ∼65% envelope protein, the remaining mass being accounted for largely by the peptidoglycan-lipoprotein structure. In this form the protein was completely resistant to trypsin, but on dissocn. it was quickly degraded to small fragments. Unlike the dissocd. polypeptide, the complexed form of the protein did not bind dodecyl sulfate tightly even on prolonged exposure to high excess at 60°. A large fraction of the polypeptide existed as β-structure, as detd. by CD and ir spectroscopy. The 105 copies of this polypeptide/cell were arranged in a lattice structure with hexagonal symmetry and a periodicity of 7.5 nm on the outer face of the peptidoglycan. The regular array obsd. appeared closely related to its quaternary structure in vivo. All strains of E. coli tested contained this protein.
- 29Sondermann, M.; George, M.; Fertig, N.; Behrends, J. C. High-resolution electrophysiology on a chip: transient dynamics of alamethicin channel formation. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 545– 551, DOI: 10.1016/j.bbamem.2006.03.023[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltFCrtrs%253D&md5=2537e69ae1f36f93bc8155f950a99a36High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formationSondermann, Markus; George, Michael; Fertig, Niels; Behrends, Jan C.Biochimica et Biophysica Acta, Biomembranes (2006), 1758 (4), 545-551CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, the authors further explore this technol. with respect to high-resoln., low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, the authors recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With the authors' setup, they were able to distinctly resolve dwell times well below 100 μs and to perform a thorough statistical anal. of alamethicin gating. The authors' results show good agreement with models that do not rely on the existence of nonconducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future expts. on cellular ion channels reconstituted in suspended lipid membranes.
Cited By
This article is cited by 5 publications.
- Essraa A. Hussein, Ryan J. White. Silver Nanoneedle Probes Enable Sustained DC Current, Single-Channel Resistive Pulse Nanopore Sensing. Analytical Chemistry 2021, 93 (33) , 11568-11575. https://doi.org/10.1021/acs.analchem.1c02087
- Jiajun Wang, Niels Fertig, Yi-Lun Ying. Real-time monitoring β-lactam/β-lactamase inhibitor (BL/BLI) mixture towards the bacteria porin pathway at single molecule level. Analytical and Bioanalytical Chemistry 2019, 411 (19) , 4831-4837. https://doi.org/10.1007/s00216-019-01650-3
- Jiajun Wang, Jayesh Arun Bafna, Satya Prathyusha Bhamidimarri, Mathias Winterhalter. Permeation von kleinen Molekülen durch Membrankanäle: Chemische Modifikation zur Quantifizierung des Transports über OmpF. Angewandte Chemie 2019, 131 (14) , 4788-4792. https://doi.org/10.1002/ange.201814489
- Jiajun Wang, Jayesh Arun Bafna, Satya Prathyusha Bhamidimarri, Mathias Winterhalter. Small‐Molecule Permeation across Membrane Channels: Chemical Modification to Quantify Transport across OmpF. Angewandte Chemie International Edition 2019, 58 (14) , 4737-4741. https://doi.org/10.1002/anie.201814489
- Muriel Masi, Mathias Winterhalter, Jean-Marie Pagès. Outer Membrane Porins. 2019, 79-123. https://doi.org/10.1007/978-3-030-18768-2_4
Abstract
Figure 1
Figure 1. Schematic diagram of the chip configuration on the Port-a-patch. (A) An agarose salt bridge has been used together with the ground electrode. A salt bridge filled with 1.5% agarose is used to cover the ground electrode to eliminate the polarization. (B) A typical GUV has been captured on a borosilicate chip forming the bilayer.
Figure 2
Figure 2. SEM characterization of the microcapillary pulled tip. The pipettes were sputtered with Au for 20 s before SEM characterizations (figure kindly provided by Long’s group, East China University of Science and Technology, Shanghai, China).
Figure 3
Figure 3. (A) Schematic diagram of the whole GUV patch clamp. (B) A vesicle got captured at the microcapillary tip. (C) An unbalanced osmotic pressure causes the vesicle to shrink 240 s after patching, reducing the size of the vesicle to less than 5 μm. The seal resistance exceeded gigaohm gradually. (D) BSA treatment: A vesicle formed in a BSA solution is patched with a BSA coated microcapillary shows no obvious shrinkage (see Whole GUV Patch Clamp for details).
Figure 4
Figure 4. Molecular structure of (A) sulbactam (MW = 233) and (B) ceftazidime (MW = 546).
References
ARTICLE SECTIONSThis article references 29 other publications.
- 1Mortimer, P. G.; Piddok, L. J. The accumulation of five antibacterial agents in porin-deficient mutants of Escherichia coli. J. Antimicrob. Chemother. 1993, 32, 195– 213, DOI: 10.1093/jac/32.2.195[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXms1Khurc%253D&md5=72a47ea868a250c0f3a59a16bdf778b7The accumulation of five antibacterial agents in porin-deficient mutants of Escherichia coliMortimer, Peter G. S.; Piddock, Laura J. V.Journal of Antimicrobial Chemotherapy (1993), 32 (2), 195-213CODEN: JACHDX; ISSN:0305-7453.A library of isogenic mutants contg. Mud9-induced deletions of the structural and regulatory genes for the porin proteins OmpF and OmpC of E. coli was constructed. The accumulation of norfloxacin, tetracycline, chloramphenicol, cephalothin, and cefoxitin was measured with each strain and shown to be reproducible with low exptl. std. deviations, such that the roles of OmpF, OmpC, and PhoE in the accumulation of these agents were detd. The loss of OmpF reduced accumulation of norfloxacin, tetracycline, cephalothin, and cefoxitin by 16-60% compared to the wild-type parent strain, but reduced accumulation of chloramphenicol by <10%. The loss of OmpC reduced accumulation of cephalothin and cefoxitin by 13 and 34%, resp., compared to the wild-type parent strain, but had little effect on the accumulation of norfloxacin, chloramphenicol, and tetracycline (<3%). The loss of both OmpF and OmpC (ompR) reduced accumulation of norfloxacin, chloramphenicol, tetracycline, cephalothin, and cefoxitin by 36-68%. However, the presence of PhoE in the absence of both OmpF and OmpC enhanced accumulation of these 5 agents to 52-119% of the concns. accumulated by the wild-type strain. These data suggest that OmpF is the preferred route of entry for 3 of the antibiotics studied, but not for chloramphenicol and tetracycline, which utilize both porins equally well. The high levels of accumulation (30-64%) of all 5 antibiotics in the absence of all major porins suggest that an alternative mechanism(s) of accumulation is available.
- 2Cowan, S. W.; Garavito, R.; Jansonius, J.; Jenkins, J.; Karlsson, R.; König, N.; Pai, E.; Pauptit, R.; Rizkallah, P.; Rosenbusch, J. The structure of OmpF porin in a tetragonal crystal form. Structure 1995, 3, 1041– 1050, DOI: 10.1016/S0969-2126(01)00240-4[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptVagtr8%253D&md5=5bd39aec8d5ef8c06336cb0b617950b6The structure of OmpF porin in a tetragonal crystal formCowan, S. W.; Garavito, R. M.; Jansonius, J. N.; Jenkins, J. A.; Karlsson, R.; Koenig, N.; Pai, E. F.; Pauptit, R. A.; Rizkallah, P. J.; et al.Structure (London) (1995), 3 (10), 1041-50CODEN: STRUE6; ISSN:0969-2126. (Current Biology)OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic mols., such as nutrients and waste products, across the outer membrane of Escherichia coli. Very few membrane proteins have been crystd. in three dimensions, yet this stable protein can be obtained in several crystal forms. Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows the authors to explore the integrity of the structure outside the natural membrane environment. The structure of OmpF porin in a tetragonal crystal form with two trimers per asym. unit has been detd. at 3.2 Å resoln. and compared with that obtained previously in a trigonal crystal form. The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice. The trimer structure is virtually identical in both. This comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints, and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided that starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.
- 3Cowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R.; Jansonius, J.; Rosenbusch, J. Crystal structures explain functional properties of two E. coli porins. Nature 1992, 358, 727– 733, DOI: 10.1038/358727a0[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XlsVGltbo%253D&md5=64f9b453e89092fb7edb57fc07bb2332Crystal structures explain functional properties of two E. coli porinsCowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R. A.; Jansonius, J. N.; Rosenbusch, J. P.Nature (London, United Kingdom) (1992), 358 (6389), 727-33CODEN: NATUAS; ISSN:0028-0836.Porins form aq. channels that aid the diffusion of small hydrophilic mols. across the outer membrane of Gram-neg. bacteria. The crystal structures of matrix porin and phosphoporin both reveal trimers of identical subunits, each subunit consisting of a 16-stranded anti-parallel β-barrel contg. a pore. A long loop inside the barrel contributes to a constriction of the channel where the charge distribution affects ion selectivity. The structures explain at the mol. level functional characteristics and their alterations by known mutations.
- 4Benz, R.; Schmid, A.; Hancock, R. Ion selectivity of gram-negative bacterial porins. J. Bacteriol. 1985, 162, 722– 727[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXitFKquro%253D&md5=545bea1d2bc6247febcba25ab9f07e90Ion selectivity of gram-negative bacterial porinsBenz, Roland; Schmid, Angela; Hancock, Robert E. W.Journal of Bacteriology (1985), 162 (2), 722-7CODEN: JOBAAY; ISSN:0021-9193.Twelve different porins from the gram-neg. bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single channel conductance between 1.5 and 6 nS in 1M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free soln. Thus, the single-channel conductances of the individual porins could be used to est. the effective channel diams. of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes contg. individual porins gave results that were consistent with the conclusions drawn from the single-channel expts. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aq. phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examd., including all of the known constitutive porins of the 4 gram-neg. bacteria studied, were cation selective with a 3-40-fold preference for K+ over Cl-.
- 5Nikaido, H. Molecular Basis of Bacterial Outer Membrane Permeability Revisited. Microbiol. Mol. Biol. Rev. 2003, 67, 593– 656, DOI: 10.1128/MMBR.67.4.593-656.2003[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmt1Sqsg%253D%253D&md5=6b2e709a18bc697e20070aafb412b06fMolecular basis of bacterial outer membrane permeability revisitedNikaido, HiroshiMicrobiology and Molecular Biology Reviews (2003), 67 (4), 593-656CODEN: MMBRF7; ISSN:1092-2172. (American Society for Microbiology)A review. Gram-neg. bacteria characteristically are surrounded by an addnl. membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compds. and at the same time to allow the influx of nutrient mols. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in mol. detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide phospholipid asym. bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compds., owing to our increasing knowledge about the chem. of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.
- 6Liu, X.; Ferenci, T. An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation. Microbiology 2001, 147, 2981– 2989, DOI: 10.1099/00221287-147-11-2981
- 7Philipsen, M. H.; Phan, N. T.; Fletcher, J. S.; Malmberg, P.; Ewing, A. G. Mass Spectrometry Imaging Shows Cocaine and Methylphenidate have Opposite Effects on Major Lipids in Drosophila Brain. ACS Chem. Neurosci. 2018, 1462– 1468, DOI: 10.1021/acschemneuro.8b00046[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjvVKrtLc%253D&md5=adfbcf34552d90d0947438ae6e1ada85Mass Spectrometry Imaging Shows Cocaine and Methylphenidate Have Opposite Effects on Major Lipids in Drosophila BrainPhilipsen, Mai H.; Phan, Nhu T. N.; Fletcher, John S.; Malmberg, Per; Ewing, Andrew G.ACS Chemical Neuroscience (2018), 9 (6), 1462-1468CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to study the effects of cocaine vs. methylphenidate administration on both the localization and abundance of lipids in Drosophila melanogaster brain. A J105 ToF-SIMS with a 40 keV gas cluster primary ion source enabled the authors to probe mol. ions of biomols. on the fly with a spatial resoln. of ∼3 μm, giving the authors unique insights into the effect of these drugs on mol. lipids in the nervous system. Significant changes in phospholipid compn. were obsd. in the central brain for both. Principal components image anal. revealed that changes occurred mainly for phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols. When the lipid changes caused by cocaine were compared with those induced by methylphenidate, it was shown that these drugs exert opposite effects on the brain lipid structure. The authors speculate that this might relate to the mol. mechanism of cognition and memory.
- 8Ghai, I.; Pira, A.; Scorciapino, M. A.; Bodrenko, I.; Benier, L.; Ceccarelli, M.; Winterhalter, M.; Wagner, R. General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpF. J. Phys. Chem. Lett. 2017, 8, 1295– 1301, DOI: 10.1021/acs.jpclett.7b00062[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVCmtbo%253D&md5=1cb3e491a3bb31fa660a8aae0d2891c1General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpFGhai, Ishan; Pira, Alessandro; Scorciapino, Mariano Andrea; Bodrenko, Igor; Benier, Lorraine; Ceccarelli, Matteo; Winterhalter, Mathias; Wagner, RichardJournal of Physical Chemistry Letters (2017), 8 (6), 1295-1301CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)A major challenge in the discovery of the new antibiotics against Gram-neg. bacteria is to achieve sufficiently fast permeation to avoid high doses causing toxic side effects. So far, suitable assays for quantifying the uptake of charged antibiotics into bacteria are lacking. The authors apply electrophysiol. zero-current assay using concn. gradients of β-lactamase inhibitors combined with single channel conductance to quantify their flux rates through OmpF. Mol. dynamic simulations provide in addn. details on the interactions between the nanopore wall and the charged solutes. In particular, the interaction barrier for three β-lactamase inhibitors is surprisingly as low as 3-5 kcal/mol, and only slightly above the diffusion barrier of ions such as chloride. Within the authors' macroscopic const. field model, the authors det. that at zero-membrane potential a concn. gradient of 10 μM of avibactam, sulbactam or tazobactam can create a flux rates of roughly 620 mols./s per OmpF trimer.
- 9Ghai, I.; Winterhalter, M.; Wagner, R. Probing transport of charged β-lactamase inhibitors through OmpC, a membrane channel from E. coli. Biochem. Biophys. Res. Commun. 2017, 484, 51– 55, DOI: 10.1016/j.bbrc.2017.01.076[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVKisrw%253D&md5=d1a6d0bf3d9158faca51ab394fe55049Probing transport of charged lactamase inhibitors through OmpC, a membrane channel from E. coliGhai, Ishan; Winterhalter, Mathias; Wagner, RichardBiochemical and Biophysical Research Communications (2017), 484 (1), 51-55CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)One of the major causes of antibiotic resistance in the Gram-neg. bacteria is the low permeability across the outer membrane. Currently a main bottleneck in the development of effective antibiotics is the lack of a general method to quantify permeation which would allow screening for optimal scaffolds. Here, we present a permeation assay based on conventional electrophysiol. The method mainly involves application of concn. gradients of charged mols. with different electrophoretic mobilities through a membrane channel. Thus the unbalanced flux creates an electrostatic potential which provides direct information on relative ion fluxes. The exptl. approach applied here involves measuring zero-current-potentials and the corresponding single channel conductance. For OmpC and the lactamase inhibitor avibactam at a 10 microm gradient the calcd. flux rate at Vm = 0mV was about n = 200 mols./s per OmpC single pore.
- 10Soundararajan, G.; Bhamidimarri, S. P.; Winterhalter, M. Understanding carbapenem translocation through OccD3 (OpdP) of Pseudomonas aeruginosa. ACS Chem. Biol. 2017, 12, 1656– 1664, DOI: 10.1021/acschembio.6b01150[ACS Full Text], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmsVaht7o%253D&md5=19c5c5cf63ff6e87db3bb7dae7c694d6Understanding Carbapenem Translocation through OccD3 (OpdP) of Pseudomonas aeruginosaSoundararajan, Gowrishankar; Bhamidimarri, Satya Prathyusha; Winterhalter, MathiasACS Chemical Biology (2017), 12 (6), 1656-1664CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Pseudomonas aeruginosa utilizes a plethora of substrate specific channels for the uptake of small nutrients. OccD3 (OpdP or PA4501) is an OprD like arginine uptake channel of P. aeruginosa whose role has been implicated in carbapenem uptake. To understand the mechanism of selective permeation we reconstituted single OccD3 channels in planar lipid bilayer and characterized the interaction with Imipenem and Meropenem analyzing the ion current fluctuation in presence of substrates. We performed point mutations in the constriction region of OccD3 to understand the binding and translocation of antibiotic in OccD3. By mutating 2 key residues in the substrate binding sites of OccD3 (located in the internal loop L7 and basic ladder) we emphasize the importance of these residues. We show that carbapenem antibiotics follow a similar path as arginine through the constriction zone and the basic ladder to translocate across OccD3.
- 11Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates Inc, 2001.Google ScholarThere is no corresponding record for this reference.
- 12Braeken, D.; Jans, D.; Huys, R.; Stassen, A.; Collaert, N.; Hoffman, L.; Eberle, W.; Peumans, P.; Callewaert, G. Open-cell recording of action potentials using active electrode arrays. Lab Chip 2012, 12, 4397– 4402, DOI: 10.1039/c2lc40656j[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVCqu73F&md5=a04797d31e56bd089ba5c1516bc598f3Open-cell recording of action potentials using active electrode arraysBraeken, Dries; Jans, Danny; Huys, Roeland; Stassen, Andim; Collaert, Nadine; Hoffman, Luis; Eberle, Wolfgang; Peumans, Peter; Callewaert, GeertLab on a Chip (2012), 12 (21), 4397-4402CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)The investigation of complex communication in cellular networks requires superior measurement tools than those available to date. Electrode arrays integrated onto silicon electronics are increasingly used to measure the elec. activity of cells in an automated and highly parallelized fashion, but they are restricted to recording extracellular potentials. Here, we report on an array of TiN electrodes built using std. silicon electronics for intracellular action potential recording. Intracellular access, possible at each of the 16 384 electrodes on the chip, was accomplished by local membrane electroporation using elec. stimulation with subcellular, micrometer-sized electrodes. Access to the cell interior was transient and could be tuned in duration by adapting the electroporation protocol. Intracellular sensing was found to be minimally invasive in the short and long-term, allowing consecutive intracellular recordings from the same cell over the course of days. Finally, we applied this method to investigate the effect of an ion channel blocker on cardiac elec. activity. This technique opens the door to massively parallel, long-term intracellular recording for fundamental electrophysiol. and drug screening.
- 13Abbott, J.; Ye, T.; Ham, D.; Park, H. Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology. Acc. Chem. Res. 2018, 51, 600– 608, DOI: 10.1021/acs.accounts.7b00519[ACS Full Text], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFOrsrc%253D&md5=d321cbe0a2297c60f074421d91654b67Optimizing Nanoelectrode Arrays for Scalable Intracellular ElectrophysiologyAbbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, HongkunAccounts of Chemical Research (2018), 51 (3), 600-608CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Electrode technol. for electrophysiol. has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold std. in studying the fundamental cellular processes underlying the elec. activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large no. of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiol. screening from basic neurobiol. labs. to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the std. top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive no. of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiol., and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful ref. Summing up from the recent studies, the authors will first elucidate the morphol. and assocd. elec. properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which the authors will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chem. modifications, electroporation protocols, and recording/stimulation electronics, as the authors describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. The authors will discuss this theme using a recent development from the groups as an example, where an array of ∼1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiol.
- 14Farre, C.; Stoelzle, S.; Haarmann, C.; George, M.; Brüggemann, A.; Fertig, N. Automated ion channel screening: patch clamping made easy. Expert Opin. Ther. Targets 2007, 11, 557– 565, DOI: 10.1517/14728222.11.4.557[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjt1Sqsrw%253D&md5=df83373ddd57528146cf70740fa22159Automated ion channel screening: patch clamping made easyFarre, Cecilia; Stoelzle, Sonja; Haarmann, Claudia; George, Michael; Brueggemann, Andrea; Fertig, NielsExpert Opinion on Therapeutic Targets (2007), 11 (4), 557-565CODEN: EOTTAO; ISSN:1472-8222. (Informa Healthcare)A review. Efficient high resoln. techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resoln. but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technol. meets the need of drug developers for higher throughput and facilitates new exptl. approaches in ion channel research. Specifically, Nanion's electrophysiol. workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated exptl. possibilities, such as accurate and fast ligand application, temp. control and internal soln. exchange. This article describes the chip-based patch clamp technol. and its usefulness in ion channel drug screening and academic research.
- 15Kreir, M.; Farre, C.; Beckler, M.; George, M.; Fertig, N. Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes. Lab Chip 2008, 8, 587– 595, DOI: 10.1039/b713982a[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjslyksb0%253D&md5=e8f6cb817f1133132a329c8a2404421dRapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomesKreir, Mohamed; Farre, Cecilia; Beckler, Matthias; George, Michael; Fertig, NielsLab on a Chip (2008), 8 (4), 587-595CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp anal., like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and obsd. the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin contg. lipid bilayers is of potential for the efficient electrophysiol. characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.
- 16Mahendran, K. R.; Kreir, M.; Weingart, H.; Fertig, N.; Winterhalter, M. Permeation of Antibiotics through Escherichia coli OmpF and OmpC Porins Screening for Influx on a Single-Molecule Level. J. Biomol. Screening 2010, 15, 302– 307, DOI: 10.1177/1087057109357791[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFWjs74%253D&md5=6f51572d413a079fdff09182751b8072Permeation of antibiotics through Escherichia coli OmpF and OmpC porins: screening for influx on a single-molecule levelMahendran, Kozhinjampara R.; Kreir, Mohamed; Weingart, Helge; Fertig, Niels; Winterhalter, MathiasJournal of Biomolecular Screening (2010), 15 (3), 302-307CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)A chip-based automated patch-clamp technique provides an attractive biophys. tool to quantify solute permeation through membrane channels. Proteo-giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics-cephalosporins and fluoroquinolones-through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to det. the mode of permeation. The chip-based setup allows rapid soln. exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-mol. level.
- 17Ionescu, S. A.; Lee, S.; Housden, N. G.; Kaminska, R.; Kleanthous, C.; Bayley, H. Orientation of the OmpF Porin in Planar Lipid Bilayers. ChemBioChem 2017, 18, 554– 562, DOI: 10.1002/cbic.201600644
- 18Gornall, J. L.; Mahendran, K. R.; Pambos, O. J.; Steinbock, L. J.; Otto, O.; Chimerel, C.; Winterhalter, M.; Keyser, U. F. Simple reconstitution of protein pores in nano lipid bilayers. Nano Lett. 2011, 11, 3334– 3340, DOI: 10.1021/nl201707d[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptVKrt7g%253D&md5=88a56c5100b6a12a732116076299f6dcSimple Reconstitution of Protein Pores in Nano Lipid BilayersGornall, Joanne L.; Mahendran, Kozhinjampara R.; Pambos, Oliver J.; Steinbock, Lorenz J.; Otto, Oliver; Chimerel, Catalin; Winterhalter, Mathias; Keyser, Ulrich F.Nano Letters (2011), 11 (8), 3334-3340CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors developed a new, simple and robust approach for rapid screening of single mol. interactions with protein channels. The authors' glass nanopipets can be fabricated simply by drawing glass capillaries in a std. pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ∼140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by elec. and mech. forces. The authors demonstrate the functional reconstitution of the Escherichia coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-mol. level, demonstrating the ability of this method to detect single mol. interactions with protein channels. High-resoln. conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-mol. studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.
- 19Garten, M.; Mosgaard, L. D.; Bornschlögl, T.; Dieudonné, S.; Bassereau, P.; Toombes, G. E. Whole-GUV patch-clamping. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 328– 333, DOI: 10.1073/pnas.1609142114[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitFentrnK&md5=757fba9d98f5630604eb235709f0de83Whole-GUV patch-clampingGarten, Matthias; Mosgaard, Lars D.; Bornschlogl, Thomas; Dieudonne, Stephane; Bassereau, Patricia; Toombes, Gilman E. S.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (2), 328-333CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiol. high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiol., but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipet and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipet. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipet surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compns. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiol. range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane compn., tension, and shape.
- 20Su, J.; Zhao, Y.; Fang, C.; Shi, Y. Asymmetric osmotic water permeation through a vesicle membrane. J. Chem. Phys. 2017, 146, 204902 DOI: 10.1063/1.4983749[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotlWitLk%253D&md5=772b89cc302acf861ac23f0c08fd7059Asymmetric osmotic water permeation through a vesicle membraneSu, Jiaye; Zhao, Yunzhen; Fang, Chang; Shi, YueJournal of Chemical Physics (2017), 146 (20), 204902/1-204902/7CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Understanding water permeation through a cell membrane is of primary importance for biol. activities and a key step to capture its shape transformation in salt soln. Here, we reveal the dynamical behaviors of osmotically driven transport of water mols. across a vesicle membrane by mol. dynamics simulations. Of particular interest is that the water transport in and out of vesicles is highly distinguishable given the osmotic forces are the same, suggesting an asym. osmotic transportation. This asym. phenomenon exists in a broad range of parameter space such as the salt concn., temp., and vesicle size, and can be ascribed to the similar asym. potential energy of lipid-ion, lipid-water, lipid-soln., lipid-lipid, and the lipid-lipid energy fluctuations. Specifically, the water flux has a linear increase with the salt concn., similar to the prediction by Nernst-Planck equation or Fick's 1st law. Furthermore, due to the Arrhenius relation between the membrane permeability and temp., the water flux also exhibits excellent Arrhenius dependence on the temp. Meanwhile, the water flux shows a linear increase with the vesicle surface area since the flux amt. across a unit membrane area should be a const. Finally, we also present the anonymous diffusion behaviors for the vesicle itself, where transitions from normal diffusion at short times to subdiffusion at long times are identified. These results provide significant new phys. insights for osmotic water permeation through a vesicle membrane and are helpful for future exptl. studies. (c) 2017 American Institute of Physics.
- 21Su, J.; Yao, Z.; de la Cruz, M. O. Vesicle Geometries Enabled by Dynamically Trapped States. ACS Nano 2016, 10, 2287– 2294, DOI: 10.1021/acsnano.5b06991[ACS Full Text], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Khsbc%253D&md5=943c6f3d33295ec34564244abf7dd97cVesicle Geometries Enabled by Dynamically Trapped StatesSu, Jiaye; Yao, Zhenwei; Olvera de la Cruz, MonicaACS Nano (2016), 10 (2), 2287-2294CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The authors design dynamic protocols for enlarging the shape space of both fluid and cryst. vesicles beyond the equil. zone. By removing water from within the vesicle at different rates, the authors numerically produced a series of dynamically trapped stable vesicle shapes for both fluid and cryst. vesicles in a highly controllable fashion. In cryst. vesicles that are continuously dehydrated, simulations show the initial appearance of small flat areas over the surface of the vesicles that ultimately merge to form fewer flat faces. In this way, the vesicles transform from a fullerene-like shape into various faceted polyhedrons. The authors perform anal. elasticity anal. to show that these salient features are attributable to the cryst. nature of the vesicle. The potential to use dynamic protocols, such as those used in this study, to engineer vesicle shape transformations is helpful for exploiting the richness of vesicle geometries for desired applications.
- 22Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques. Biochim. Biophys. Acta, Biomembr. 1996, 1279, 169– 180, DOI: 10.1016/0005-2736(95)00274-X[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhsFOjtbc%253D&md5=f62efc4a6b801c312e9ebef4e6951cbcImpedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniquesSteinem, Claudia; Janshoff, Andreas; Ulrich, Wolf-Peter; Sieber, Manfred; Galla, Hans-JoachimBiochimica et Biophysica Acta, Biomembranes (1996), 1279 (2), 169-80CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)One topic of this study is the comparison of different prepn. techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid soln. in n-decane/isobutanol, (iii) by the lipid/detergent diln. technique or (i.v.) by fusion of vesicles. (2) Charged mols. carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Neg. charged surfaces of 3-mercaptopropionic acid were excellent substrates that allow the attachment of planar lipid bilayers by applying pos. charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or neg. charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If pos. charged first monolayers of mercaptoethylammoniumhydrochloride were used the authors were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles contg. 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A crit. amt. of more than 50 mol% of DMPTE was necessary to form a solid supported lipid bilayer. Bilayers obtained with these different prepn. techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. The authors used a DODAB-bilayer for the reconstitution of gramicidin D channels. By CD measurements the authors verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance anal.
- 23Benz, R.; Schmid, A.; Nakae, T.; Vos-Scheperkeuter, G. Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J. Bacteriol. 1986, 165, 978– 986, DOI: 10.1128/jb.165.3.978-986.1986[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XhsVGhs7s%253D&md5=34eb9b2235e3f4a53426e6aad938bd00Pore formation by LamB of Escherichia coli in lipid bilayer membranesBenz, Roland; Schmid, Angela; Nakae, Taiji; Vos-Scheperkeuter, Greetje H.Journal of Bacteriology (1986), 165 (3), 978-86CODEN: JOBAAY; ISSN:0021-9193.Lipid bilayer expts. were performed in the presence of different E. coli LamB prepns. These LamB prepns. formed 2 types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised ∼1-6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1M KCl. These pores could be completely blocked by the addn. of maltose and maltodextrins. Titrn. of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 × 10-4M for maltotriose. Apparently, the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.
- 24Bhamidimarri, S. P.; Prajapati, J. D.; van den Berg, B.; Kleinekathoefer, U.; Winterhalter, M. Electro-Osmotic Driven Kinetics of Cyclodextrin through the CymA Channel. Biophys. J. 2016, 110, 115a, DOI: 10.1016/j.bpj.2015.11.673
- 25Bhamidimarri, S. P.; Prajapati, J. D.; van den Berg, B.; Winterhalter, M.; Kleinekathöfer, U. Role of electroosmosis in the permeation of neutral molecules: CymA and cyclodextrin as an example. Biophys. J. 2016, 110, 600– 611, DOI: 10.1016/j.bpj.2015.12.027[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnvFGrtQ%253D%253D&md5=4c1ed5c0adb3525daf20b501100163f1Role of Electroosmosis in the Permeation of Neutral Molecules: CymA and Cyclodextrin as an ExampleBhamidimarri, Satya Prathyusha; Prajapati, Jigneshkumar Dahyabhai; van den Berg, Bert; Winterhalter, Mathias; Kleinekathoefer, UlrichBiophysical Journal (2016), 110 (3), 600-611CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)To quantify the flow of small uncharged mols. into and across nanopores, one often uses ion currents. The resp. ion-current fluctuations caused by the presence of the analyte make it possible to draw some conclusions about the direction and magnitude of the analyte flow. However, often this flow appears to be asym. with respect to the applied voltage. As a possible reason for this asymmetry, the authors identified the electroosmotic flow (EOF), which is the water transport assocd. with ions driven by the external transmembrane voltage. As an example, the authors quantify the contribution of the EOF through a nanopore by studying the permeation of α-cyclodextrin through CymA, a cyclodextrin-specific channel from Klebsiella oxytoca. To understand the results from electrophysiol. on a mol. level, all-atom mol. dynamics simulations were used to detail the effect of the EOF on substrate entry to and exit from a CymA channel in which the N-terminus has been deleted. The combined exptl. and computational results strongly suggest that one needs to account for the significant contribution of the EOF when analyzing the penetration of cyclodextrins through the CymA pore. This example study at the same time points to the more general finding that the EOF needs to be considered in translocation studies of neutral mols. and, at least in many cases, should be able to help in discriminating between translocation and binding events.
- 26Guzel, F. D.; Citak, F. Development of an On-Chip Antibiotic Permeability Assay With Single Molecule Detection Capability. IEEE Trans. Nanobiosci. 2018, 17, 155– 160, DOI: 10.1109/TNB.2018.2809592
- 27Saha, S. C.; Powl, A. M.; Wallace, B. A.; de Planque, M. R.; Morgan, H. Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip. Biomicrofluidics 2015, 9, 014103 DOI: 10.1063/1.4905313[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslCnug%253D%253D&md5=54bd1e95233b8967d3dbee7be20e1361Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chipSaha, Shimul C.; Powl, Andrew M.; Wallace, B. A.; de Planque, Maurits R. R.; Morgan, HywelBiomicrofluidics (2015), 9 (1), 014103/1-014103/11CODEN: BIOMGB; ISSN:1932-1058. (American Institute of Physics)We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiol. screening of ion channels and transporters. A passive pumping method is used to flow microliter vols. of ligand soln. across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 μl/min. Phospholipid bilayers are formed across a photolithog. defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with β-cyclodextrin (β-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for detg. IC50 values. The assays are fast (30 min for a complete IC50 curve) and simple and require very small amts. of compds. (100 μg in 15 μl). The technol. can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores. (c) 2015 American Institute of Physics.
- 28Rosenbusch, J. P. Characterization of the major envelope protein from Escherichia coli regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J. Biol. Chem. 1974, 249, 8019– 8029[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2MXmvFCmtw%253D%253D&md5=cd26fa2c682ad0570d0058f2d42a78afCharacterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate bindingRosenbusch, Jurg P.Journal of Biological Chemistry (1974), 249 (24), 8019-29CODEN: JBCHA3; ISSN:0021-9258.The major envelope protein from E. coli was purified by differential heat extn. in dodecyl sulfate and subsequently freed of the detergent. The polypeptide was homogenous and had a mass of 36,500 daltons. Homogeneity was based on 4 criteria, 3 of which were independent of its behavior in detergents. The mass of the protein was accounted for entirely, or nearly entirely, by the mass of its constituent amino acids. Probably, dodecyl sulfate is bound in amts. corresponding to those found in most polypeptides. The protein was also isolated in assocn. with the rigid layer of the cell by extn. of cell envelopes in 2% dodecyl sulfate at 60°. This complex was composed of ∼65% envelope protein, the remaining mass being accounted for largely by the peptidoglycan-lipoprotein structure. In this form the protein was completely resistant to trypsin, but on dissocn. it was quickly degraded to small fragments. Unlike the dissocd. polypeptide, the complexed form of the protein did not bind dodecyl sulfate tightly even on prolonged exposure to high excess at 60°. A large fraction of the polypeptide existed as β-structure, as detd. by CD and ir spectroscopy. The 105 copies of this polypeptide/cell were arranged in a lattice structure with hexagonal symmetry and a periodicity of 7.5 nm on the outer face of the peptidoglycan. The regular array obsd. appeared closely related to its quaternary structure in vivo. All strains of E. coli tested contained this protein.
- 29Sondermann, M.; George, M.; Fertig, N.; Behrends, J. C. High-resolution electrophysiology on a chip: transient dynamics of alamethicin channel formation. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 545– 551, DOI: 10.1016/j.bbamem.2006.03.023[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltFCrtrs%253D&md5=2537e69ae1f36f93bc8155f950a99a36High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formationSondermann, Markus; George, Michael; Fertig, Niels; Behrends, Jan C.Biochimica et Biophysica Acta, Biomembranes (2006), 1758 (4), 545-551CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, the authors further explore this technol. with respect to high-resoln., low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, the authors recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With the authors' setup, they were able to distinctly resolve dwell times well below 100 μs and to perform a thorough statistical anal. of alamethicin gating. The authors' results show good agreement with models that do not rely on the existence of nonconducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future expts. on cellular ion channels reconstituted in suspended lipid membranes.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01611.
Patched vesicles electrical behavior and microscopic image; pipette pulling parameters as well as the permeability calculation approaches; purification of membrane proteins (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.