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Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

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† Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, United States
‡ Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, United States
*Department of Molecular and Cellular Biology, Harvard University, 52 Oxford St., Cambridge, MA 02138. Telephone: (617) 495-5616. Fax: (617) 496-9684. E-mail: [email protected]
Cite this: Biochemistry 2012, 51, 31, 6195–6206
Publication Date (Web):June 15, 2012
https://doi.org/10.1021/bi300279b

Copyright © 2012 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

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The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

  Funding Statement

This work was funded by National Institutes of Health Grant R01GM081340 to R.G. This work is based upon research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines supported by Grant RR-15301 from the National Center for Research Resources. The Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. M.S. was a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation.

‡ Author Present Address

Howard Hughes Medical Institute and Department of Biochemistry, University of Washington, Seattle, WA 98195.

Transient receptor potential (TRP) channels are cation channels involved in sensation of various stimuli from internal and external environments. The TRP channel superfamily is divided into six subfamilies in mammals: canonical or classical TRPC, vanilloid TRPV, melastatin TRPM, ankyrin TRPA, mucolipin TRPML, and polycystin TRPP. (1-3) Six TRPV proteins, TRPV1–6, belong to the vanilloid subfamily. TRPV proteins function as tetramers, and each protomer contains six transmembrane segments flanked by two intracellular domains: a large N-terminal domain containing ankyrin repeats and a short C-terminal domain.
TRPV channel activity is regulated by post-translational modifications such as phosphorylation or binding of regulatory molecules to intracellular domains. (4, 5) In TRPV1, for example, ATP and calmodulin (CaM) have been shown to bind to the N-terminal ankyrin repeat domain (ARD) and sensitize and desensitize channel activity, respectively. (6, 7) The binding of ATP and CaM to the ARD is also conserved in TRPV4 and TRPV3. (8) Phosphatidylinositol 4,5-bisphosphate (PIP2) and CaM interact with the C-terminal domain of TRPV1 and regulate channel activity. (9-11) The molecular mechanisms by which these intracellular signals regulate TRPV channel activity remain unclear.
TRPV4 is a member of the TRPV subfamily expressed broadly in neuronal and non-neuronal cells. TRPV4 is activated by various stimuli, including hypo-osmolarity, warm temperature, and chemical ligands such as 4-α-phorbol esters and epoxyeicosatrienoic acids. (12-15) Consistent with these in vitro findings, TRPV4 has been implicated in physiological functions such as osmoregulation and thermoregulation. (16, 17) TRPV4 also plays a role in mechanosensation in the vascular endothelium and unitary tract, (18) and cell barrier formation in vascular and epidermal tissues. (19) Recently, studies using TRPV4 knockout mice suggested the functional importance of TRPV4 in the central nervous system, nociception, and bone formation. (20-22)
Mutations in TRPV4 are associated with a wide spectrum of inherited diseases, primarily autosomal dominant neuropathies and skeletal dysplasias. (23, 24) Several mutants studied in heterologous expression systems showed constitutive basal activity and enhanced response to stimuli. (25-28) These findings are consistent with cell death caused by high calcium influx such as the observed degeneration of motor neurons in neuropathies. (27, 28) However, it is difficult to explain how seemingly similar molecular properties lead to such diverse disease phenotypes. It is therefore essential to elucidate how TRPV4 is regulated at the molecular level to understand the mechanisms behind these inherited diseases.
To obtain insight into regulatory mechanisms of TRPV4, we focused on TRPV4-ARD, which can mediate channel regulation through binding of ligands to its concave surface and is the target of many mutations causing human diseases. We determined the structures of ATP-free and -bound human TRPV4-ARD and compared them with the structures of the other TRPV-ARDs. Despite a number of TRPV-ARD structures available in the absence or presence of ATP, this represents the first cognate pair of ATP-free and -bound structures. Structural analysis revealed that a long loop forming Finger 3 is flexible and could switch conformation to possibly regulate channel activity. Comparisons of TRPV-ARD structures provide further insights into the evolution of ATP binding in TRPV channels. Thermal stability analyses, a cysteine accessibility assay, and molecular dynamics (MD) simulations indicate that ATP binding can stabilize TRPV1- and TRPV4-ARD. Interestingly, most mutations causing human genetic diseases in TRPV4-ARD are located away from the ATP-binding site, suggesting that additional regulatory interactions and mechanisms exist. A series of biochemical analyses of disease-associated mutants showed that some impaired thermal stability and/or ATP binding ability, providing biochemical insights into possible disease mechanisms.

Experimental Procedures

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Expression Constructs

Human TRPV4 cDNA was obtained from C. J. Sumner. The DNA fragment corresponding to the ARD (residues 149–396) was amplified by polymerase chain reaction and subcloned into the NdeI and NotI sites of a pET vector with a C-terminal six-histidine tag (pET21-C6H). (29) Mutations were generated with QuikChange (Stratagene) and confirmed by DNA sequencing.

Protein Production and Purification

Recombinant human TRPV4-ARD protein was expressed in Escherichia coli BL21(DE3) by induction with 75 μM isopropyl β-d-thiogalactopyranoside for 13 h at room temperature after the cells reached an OD600 of ∼0.6. Frozen cell pellets were thawed, resuspended, and lysed by sonication in lysis buffer [20 mM Tris-HCl, 300 mM NaCl, and 20 mM imidazole (pH 7.0)] with 1 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1% Triton X-100, 0.2 mg/mL lysozyme, 50 μg/mL RNase A, and 25 μg/mL DNase I. The lysate was cleared by centrifugation and the supernatant loaded onto nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) and eluted with a step gradient containing 25, 50, 100, 150, 200, and 250 mM imidazole (pH 7.0) in lysis buffer with 0.05% β-mercaptoethanol and 0.5 mM PMSF. Fractions containing the protein at 150, 200, and 250 mM imidazole were combined, and EDTA (pH 8.0) was added to a final concentration of 1 mM and then the mixture dialyzed against 20 mM Tris-HCl (pH 7.0), 50 mM NaCl, 1 mM EDTA, and 1 mM DTT. The dialyzed protein was purified on SP Sepharose FF (GE Healthcare, Piscataway, NJ) in 20 mM Tris-HCl (pH 7.0), 1 mM EDTA, and 1 mM DTT using a linear gradient from 0 to 0.4 M NaCl. Size exclusion chromatography on a Superdex 75 column (GE Healthcare) in 10 mM Tris-HCl (pH 7.0), 300 mM NaCl, 10% glycerol, and 1 mM DTT was used for final purification. Protein was concentrated to ∼8 mg/mL in a Vivaspin centrifugal filter (10000 molecular weight cutoff; Sartorius AG, Goettingen, Germany), flash-frozen, and stored at −80 °C. In the purification of mutant proteins for the thermal stability assay, dialysis and ion exchange steps were omitted. After Ni-NTA purification, the proteins were purified by size exclusion chromatography in phosphate-buffered saline (pH 7.4) with 1 mM DTT. Rat TRPV1-ARD (rTRPV1-ARD) was purified as described previously. (7)

Crystallization of Human TRPV4-ARD

Crystals were grown by hanging-drop vapor diffusion at 4 °C: the ATP-free form with a 1:3 ratio of protein to reservoir solution [0.35 M NaH2PO4, 0.35 M KH2PO4, 10% glycerol, and 0.1 M Na-HEPES (pH 7.8)] and the ATP-bound form with a 1:1 ratio of protein to reservoir [3% PEG 4000, 10% glycerol, and 0.1 M Na-HEPES (pH 7.9)] in the presence of 5 mM ATP. Crystals were soaked in reservoir solution containing 15% glucose for 3 min, transferred into reservoir solution containing 30% glucose, and flash-frozen in liquid nitrogen.

Data Collection, Structure Determination, and Analysis

X-ray data were collected at 100 K using an ADSC Q315 detector at the Advanced Photon Source ID24 beamline, processed in HKL2000; (30) data statistics are listed in Table 1. The hTRPV4-ARD structures were determined by molecular replacement using the chicken TRPV4-ARD structure in MOLREP (31) and Phaser. (32) Model building was performed in COOT (33) and refinement with TLS (Translation/Libration/Screw) in REFMAC5. (34) Final refinement statistics are listed in Table 1. The coordinates have been deposited in the Protein Data Bank (PDB) as entries 4DX1 (ATP-free) and 4DX2 (ATP-bound). Figures were generated with PyMOL (Schrödinger, LLC).
Table 1. Data Collection and Refinement Statistics
 crystal form I (ATP-free)crystal form II (with ATP)
Data Collection
space groupP3121P3221
wavelength (Å)0.979170.97917
cell dimensions a, b, c (Å)53.30, 53.37, 440.71147.89, 147.89, 93.90
resolution (Å)a40.0–2.85(2.90–2.85)40.0–2.95(3.00–2.95)
Rsyma0.115 (0.614)0.099 (0.714)
I/σ(I)a12.3 (1.8)17.6 (2.4)
completeness (%)a99.6 (99.3)100.0 (100.0)
redundancya5.3. (5.4)5.5 (5.6)
Refinement
resolution (Å)a39.2–2.85(2.92–2.85)38.86–2.95(3.03–2.95)
no. of reflections1730623958
Rwork/Rfree0.215/0.2780.177/0.220
no. of molecules per asymmetric unit22
no. of residues in model148–397(chain A)148–394 (ATP-bound)
 148–396(chain B)148–392 (ATP-unbound)
no. of atoms  
protein1995 (chain A)1987 (ATP-bound)
 1986 (chain B)1963 (ATP-unbound)
ligand25 (PO4)31 (ATP)
 6 (glycerol)6 (glycerol)
  12 (glucose)
water1872
B factor (Å)  
protein80.9162.12 (ATP-bound)
  72.76 (ATP-unbound)
ligand113.98 (PO4)91.00 (ATP)
 103.44 (glycerol)72.51 (glycerol)
  90.86 (glucose)
water76.0654.84
rmsd  
bond lengths (Å)0.0130.013
bond angles (deg)1.511.80
a

Values from the highest-resolution shell are in parentheses.

Thermal Stability Assay by Circular Dichroism Spectroscopy

Circular dichroism (CD) spectra were measured at 10 °C with 3.4 μM protein in 150 mM NaCl, 5 mM Tris-HCl (pH 7.5), and 1 mM DTT for experiments testing the influence of ATP on protein stability, and in phosphate-buffered saline (pH 7.4) with 1 mM DTT for analyses of the wild type and mutants. CD spectra were recorded in a 1 mm path length cuvette on a Jasco J-815 spectropolarimeter. For thermal stability, the molar ellipticity at λ = 222 nm was measured as the protein solutions were heated at a rate of 1 °C/min and used to determine the fraction of protein folded (assuming a completely folded protein population at a starting temperature of 10 °C). Thermal denaturation temperatures (Tm) were analyzed with Jasco Spectra software (JASCO Inc., Easton, MD) and are given with their standard deviation (SD).

Molecular Dynamics Simulation of rTRPV1-ARD

Two sets of molecular dynamics simulations (apo and ATP) were conducted using the structure of rTRPV1-ARD bound to ATP, with and without the ATP ligand removed in silico, respectively (PDB entry 2PNN, residues 111–356). Structures were solvated in explicit water (TIP3) and ions (100 mM NaCl) using VMD. (35) The systems contained 208501 (ATP) and 208482 (apo) atoms. Minimization, equilibration, and steered molecular dynamics (SMD) simulations were performed in multiple steps using NAMD2.7, (36) the CHARMM27 force field for proteins with the CMAP correction, (37, 38) full electrostatics, and 2 fs time steps with hydrogen bonds constrained. SMD simulations stretched both ends of TRPV1-ARD at effective speeds of 2 and 20 nm/ns.

ATP-Agarose Pull-Down Assays

The ATP-agarose assays were performed as described previously. (28) Briefly, 20 μg of protein was incubated with 70 or 75 μL of a 50% ATP agarose slurry for 1 h at 4 °C in binding buffer [10 mM Tris-HCl (pH 7.0), 50 mM NaCl, 1 mM DTT, and 0.15% n-decyl d-maltopyranoside]. After the agarose had been washed three times, proteins were extracted and loaded onto the 15% SDS gel. In each load lane, the volumes loaded corresponded to 1 μg of protein. Gels were quantified using ImageJ (National Institutes of Health, Bethesda, MD), and shown are the averages ± SD for three independent experiments.

TRPV1 Cysteine Modification Assay

Reaction mixtures containing rTRPV1-ARD (8.5 μM) in 150 mM NaCl, 20 mM Tris (pH 7.0), 0.5 mM PEG-maleimide 5 kDa (Creative PEGWorks), and 10 mM nucleotide as indicated were incubated at room temperature, reactions stopped by addition of DTT to a final concentration of 110 mM, and mixtures analyzed by Coomassie-stained 12% SDS–PAGE.

Statistical Analyses

Statistical significance was tested by multiple comparisons using Tukey-Kramer or Dunnett’s method with JMP software (SAS Institute Inc., Cary, NC).

Results

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Structures of Human TRPV4-ARD

Recombinant human TRPV4-ARD (hTRPV4-ARD, residues 149–396) produced in E. coli was purified for structure determination by X-ray crystallography and biochemical analyses. We determined the structure of hTRPV4-ARD in the absence (crystal form I, ATP-free) or presence (crystal form II) of ATP (Table 1). Both crystal forms contain two molecules per asymmetric unit. In crystal form II, one molecule is bound to ATP (ATP-bound) while the other is not (ATP-unbound). As expected on the basis of previous structures of TRPV-ARDs, (7) hTRPV4-ARD consists of six ankyrin repeats (ANK1–6) each containing an inner and an outer helix followed by a connecting finger loop (Fingers 1–5) (Figure 1A). The inner helices and fingers form a concave surface to which ATP is bound at the expected site previously identified in TRPV1. (7) Importantly, these structures represent the first cognate pair of ATP-bound and ATP-free TRPV-ARD structures.

Figure 1

Figure 1. Structural comparison of human and chicken TRPV4-ARDs. (A) Superimposed ribbon diagrams of ATP-bound (magenta) and ATP-free (blue) hTRPV4-ARD. ATP is shown as sticks. (B) Superimposed Cα traces of human and chicken TRPV4-ARD. Finger 3 is twisted and shrunken in the ATP-bound (magenta) and ATP-unbound (green) hTRPV4-ARD structures, while the finger is extended in ATP-free hTRPV4-ARD (blue) and cTRPV4-ARD (gray). Several Finger 3 residues are disordered in three of six TRPV4-ARD structures. The structure of Finger 2 in the ATP-bound and -unbound forms differs from that in ATP-free forms.

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The structures of hTRPV4-ARD were compared to each other and to those of chicken TRPV4-ARD (cTRPV4-ARD). (28) The structures of ATP-free hTRPV4-ARD and cTRPV4-ARD (residues 133–382, 90% identical sequences) are very similar (rmsd ranges from 0.635 to 0.922 Å over 239–242 Cα atoms) except at the tips of Fingers 2 and 3 (Figure 1B). The same trend is apparent when extending this comparison to all 10 available structures of TRPV4-ARD (four hTRPV4-ARD molecules in two crystal forms and six cTRPV4-ARD molecules in two crystal forms as well), with large structural differences observed in the very long Finger 3, as well as in the tip of Finger 2. Consistently, Finger 3 residues had high B factors and were too disordered to trace in several cTRPV4-ARD structures, indicating high flexibility.

Structural Consequences of ATP Binding

Direct comparisons of the ATP-free and ATP-bound hTRPV4-ARD structures again show conformational differences restricted to Fingers 2 and 3 (Figure 1A). Finger 3 was extended in ATP-free hTRPV4-ARD (and in ATP-free cTRPV4-ARD), while the finger loop was twisted and shrunken in ATP-bound hTRPV4-ARD. Finger 2 was also slightly twisted in ATP-bound hTRPV4-ARD. Surprisingly, both conformational differences were also observed when comparing the second, ATP-unbound, molecule in crystal form II to the ATP-free crystal form I structures (Figure 1B). In crystal form II, an adjacent symmetry-related hTRPV4-ARD molecule occupied the ATP-binding site of the ATP-unbound molecule, possibly mimicking ATP binding. Alternatively, the shrunken Finger 3 structure may result from crystal contacts and/or crystallization condition differences.
One unusual feature of TRPV-ARDs is that many aromatic residues are exposed on flexible Fingers 2 and 3. (29) In hTRPV4-ARD, 9 of 20 aromatic residues are located on the concave face of Fingers 2 and 3 [three and six residues, respectively (Figure 2)]. Interestingly, F272 and F273 on Finger 3 are exposed in the ATP-free form, while these phenylalanines are buried in the shrunken Finger 3 of the ATP-bound form (Figure 2B). The observed conformational differences may thus serve to regulate the exposure of the aromatic patch, and the conservation of the patch suggests that it is important, perhaps in interactions with regulatory ligands or with other regions of the full TRPV4 homotetramer.

Figure 2

Figure 2. Aromatic residues on Fingers 2 and 3 have varied positions in hTRPV4-ARD structures. (A) The hTRPV4-ARD structures of ATP-bound (magenta), ATP-unbound (green), and ATP-free (blue) forms are superimposed. Aromatic residues are shown as sticks. (B) Detail of the Finger 2 and 3 loops. F272 and F273 on Finger 3 (black rectangles) are embedded in the aromatic cluster in the ATP-bound and -unbound forms but exposed in the ATP-free form. Y235 and Y236 on Finger 2 and Y281 and F282 on Finger 3 are located in similar positions but show variable orientations. F231, F282, Y283, and F284 show less variation.

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Structural Comparison of TRPV-ARDs

Multiple TRPV-ARD structures are available: a structure of rat TRPV1-ARD (rTRPV1-ARD) bound to ATP, (7) structures of cTRPV4-ARD in the absence of ATP, and structures of several ARDs that do not bind ATP, namely, rat and human TRPV2-ARDs and mouse TRPV6-ARD (28, 39, 40) (see Table S1 of the Supporting Information). The overall structures of all available TRPV-ARDs are similar, with rmsd values ranging from 0.637 to 1.865 Å over 139–155 core Cα atoms (Table S2 of the Supporting Information). A superposition of all structures illustrates that the largest structural differences are again found in Fingers 2 and 3 (Figure 3A). Structural comparisons of these TRPV-ARDs and the new human TRPV4-ARD highlight three noteworthy features.

Figure 3

Figure 3. Structural comparison of TRPV-ARDs. (A) Superimposed main chain structure of TRPV-ARDs (rTRPV1-ARD, gray; rat and human TRPV2-ARD, cyan; ATP-bound hTRPV4-ARD, magenta; ATP-unbound hTRPV4-ARD, green; ATP-free hTRPV4-ARD, blue; and mouse TRPV6-ARD, black). Finger 3 and a part of Finger 2 are highly flexible. Several residues on Finger 3 are missing in one of two TRPV1-ARD structures and four of seven TRPV2-ARD structures. (B) ATP-binding site of hTRPV4-ARD and rTRPV1-ARD. Residues (sticks) within 4 Å of the ATP molecule and a surface map of the ATP-binding site in hTRPV4-ARD (left) and the corresponding residues in the rTRPV1-ARD ATP-binding site (right). The bBound ATP molecule is shown as sticks (orange and yellow). (C) Finger 2 (2) and Finger 3 (3) structures of ATP-bound rat TRPV1-ARD (gray), rat and human TRPV2-ARD (cyan), human ATP-bound TRPV4-ARD (magenta), and mouse TRPV6-ARD (black). (D) Aromatic residue positioned behind the adenine base of ATP in Finger 2 (F231 in human TRPV4-ARD). (E) This aromatic residue is conserved in TRPV-ARDs that bind ATP (red rectangle). (F and G) ATP-agarose pull-down assays for wild-type and mutant rTRPV1-ARD (F) or hTRPV4-ARD (G). Coomassie-stained gels (top) of wild-type and mutant proteins loaded (left) and bound to ATP-agarose in the absence (middle) or presence (right) of competing free ATP. The normalized intensity of protein recovered (mean ± SD; n = 3) is plotted below. The statistical significance of the change in binding to ATP-agarose with respect to the wild type (WT) was determined by a multiple-comparison test using Dunnett’s method, with p < 0.01 indicated by an asterisk.

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First, as expected from biochemical, mutational, and sequence analyses, (8) the interaction of ATP with hTRPV4-ARD is very similar to what was previously observed in rTRPV1-ARD (Figure 3B). (7) The adenine base fits in a conserved pocket, stacking against Tyr236 and hydrogen bonding to Gln239. Furthermore, Lys192 and Lys197 on the surface of inner helix 2 interact with the phosphate groups of ATP. These lysines are homologous to lysines critical for ATP binding in rTRPV1-ARD (Lys155 and Lys160) (7) and cTRPV4-ARD (Lys178 and Lys183). (8) The importance of Lys197 in ATP binding is further discussed below. The γ-phosphate of the ATP is further removed from the protein in TRPV4 compared to TRPV1, consistent with the fact that TRPV4-ARD exhibited less selectivity for ATP over ADP in ATP-agarose competition assays. (7)
Second, the backbone structure of ATP-bound hTRPV4-ARD, with a collapsed Finger 3, is quite similar to those of TRPV2-ARD and TRPV6-ARD, which lack the ability to bind ATP (Figure 3C). In contrast, the ATP-free structures of chicken and human TRPV4-ARD, which both have the ability to bind ATP, have extended Finger 3 conformations (Figures 1B and 3A). This visual observation is supported by a comparison of rmsd values for Fingers 2 and 3 of both ATP-free and -bound forms of hTRPV4-ARD with TRPV2-ARDs and mTRPV6-ARD: the ATP-bound form generally showed smaller rmsds than the ATP-free form (Table S2 of the Supporting Information). This suggests that although the ARDs of TRPV2 and TRPV6 do not bind ATP, they are closest to the ATP-bound state of ARDs with ATP-binding sites, at least in structural terms.
Finally, the structure of ATP-bound hTRPV4-ARD and comparisons to TRPV2 and TRPV6 suggest that a previously unrecognized residue, Phe231 in hTRPV4-ARD, could be important for ATP binding. Phe231 is located under the adenine-stacking Tyr236 on Finger 2 (Figure 3D), contributing to the adenine-binding pocket. This aromatic residue is conserved in the ATP-binding site of rTRPV1-ARD (Tyr194), while the aromatic residues are substituted with a smaller aliphatic residue, cysteine or methionine, in TRPV2 or TRPV6, respectively (Figure 3E). Sequence alignments of TRPV proteins confirm this trend, as the equivalent residue is a tyrosine in TRPV3, which binds ATP, and threonine in TRPV5, which does not. Substitution of these aromatic residues with cysteine (the residue found in the equivalent position in TRPV2) caused a significant reduction in the level of ATP binding in both rTRPV1-ARD and hTRPV4-ARD. In rTRPV1-ARD, the Y194C mutation reduced the level of ATP binding comparably to the K155A mutation identified in a previous study (Figure 3F). (7) A significant reduction in the level of ATP binding was also observed in hTRPV4-ARD mutant F231C (Figure 3G). These results indicate that the buried aromatic residue in Finger 2 conserved in TRPV1, TRPV3, and TRPV4 does indeed contribute to ATP binding in rTRPV1-ARD and hTRPV4-ARD.

ATP Binding Stabilizes TRPV4-ARD and TRPV1-ARD

ATP binding does not result in large conformational changes, but the ATP-bound form showed a tightly packed Finger 3 in both rTRPV1-ARD and hTRPV4-ARD structures, leading us to hypothesize that ATP may stabilize the proteins. In other ankyrin repeat proteins, such as IκBα and Notch, the ankyrin repeats are partially folded and complete their folding as a ligand is bound. (41-43) We therefore sought to determine whether ATP binding affected ARD stability. To this end, we analyzed the thermal stability of hTRPV4-ARD by CD spectroscopy in the absence or presence of ATP, AMP, or phosphate (Figure 4). In buffer alone, hTRPV4-ARD showed a melting temperature (Tm) of 37.1 ± 0.1 °C. Phosphate ions (1 mM) increased Tm slightly but not significantly (37.33 ± 0.07 °C). In the presence of nucleotide ligands (1 mM), however, a small but significant increase in Tm was observed (Tm = 37.9 ± 0.2 °C for ATP and 37.4 ± 0.2 for AMP). The greater stabilization observed with ATP compared to AMP matches their differing binding affinity observed with cTRPV4-ARD. (8)

Figure 4

Figure 4. Effect of ATP on hTRPV4-ARD thermal stability. (A) Representative circular dichroism spectra of the purified TRPV4-ARD protein (3.4 μM) in the presence of ATP, AMP, or phosphate (1 mM each) at 10 °C. The wavelength (λ) of 222 nm used for thermostability assays is indicated by a vertical red line. (B) Representative traces of the thermostability assay. The molar ellipticity at 222 nm was measured as the protein solutions were heated at a rate of 1 °C/min. (C) Tm of TRPV4-ARD in the presence of 1 mM ATP, AMP, or phosphate. The statistical significance of the change in Tm was determined by a multiple-comparison test using the Tukey–Kramer method, with p < 0.05 and p < 0.01 indicated by one asterisk and two asterisks, respectively.

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Stabilization of the ARD fold by nucleotides may also affect the accessibility of buried cysteine residues. hTRPV4-ARD contains four cysteines, two exposed (Cys194 and Cys250) and two buried (Cys294 and Cys353; Figure S1 of the Supporting Information). We examined the effect of ATP binding on ARD stability by measuring the kinetics of cysteine modification with PEG-maleimide (mPEG) in the presence or absence of 10 mM AMP or ATP. hTRPV4-ARD was modified at endogenous cysteines by a 5 kDa PEG-maleimide, resulting in multiple shifted bands on SDS–PAGE. Furthermore, the rate of protein modification with mPEG was significantly reduced in the presence of nucleotides, especially ATP (Figure S1 of the Supporting Information). This suggests that the ARD fold is quite flexible, and that ATP binding stabilizes the fold, consistent with the thermal stability results described above.
To gain further insights into the effect of ATP binding on ARD stability, we used single-cysteine mutants, allowing us to specifically address the accessibility of a buried cysteine. Interestingly, rTRPV1-ARD possesses a cysteine residue accessible to chemical modification by allicin, the active compound in garlic extract. (44) This cysteine residue, Cys157 (corresponding to Cys194 in hTRPV4-ARD), is paradoxically buried within the hydrophobic core of TRPV1-ARD (Figure 5A), suggesting that the structure may have some flexibility in solution, similar to that hypothesized for TRPV4-ARD above. Indeed, rTRPV1-ARD was modified at endogenous cysteines, like hTRPV4-ARD, whereas no mobility shift was observed in a cysteine-less rTRPV1-ARD variant (CL) (Figure 5B). More importantly, PEG-maleimide modification of a single-cysteine variant (C157) of rTRPV1-ARD, in which all cysteines except Cys157 were substituted with serine (C126S/C257S/C362S), resulted in a single shifted band (Figure 5B). This result demonstrates that buried Cys157 is indeed accessible to modification (Figure 5B), indicating a flexible and/or unstable fold. We hypothesized that ATP binding may stabilize the rTRPV1-ARD fold as it did hTRPV4-ARD. To test this hypothesis biochemically, we took advantage of the modifiable property of Cys157 to test the accessibility of this residue in the presence or absence of ATP. The single-cysteine variant rTRPV1-ARD CL-TRPV1-ARD C157 was chemically modified by PEG-maleimide with a half-time of 4 min (t1/2 = 4 min) at room temperature (Figure 5C,D). Incubation with 10 mM ATP greatly inhibited the reaction, with a t1/2 of 52 min, consistent with ATP stabilizing rTRPV1-ARD and limiting access to buried Cys157. In contrast, incubation with 10 mM ADP, a poor rTRPV1-ARD ligand, (7) only weakly inhibited Cys157 modification (t1/2 = 10 min). CL-TRPV1-ARD C362, containing a single surface-exposed cysteine in the disordered C-terminal tail of the rTRPV1-ARD construct, was rapidly modified (t1/2 < 30 s) even in the presence of nucleotides, confirming that ATP specifically inhibits the reaction of buried Cys157 (Figure 5C,D). Therefore, as shown for hTRPV4-ARD above using CD spectroscopy, cysteine modification of hTRPV4-ARD and rTRPV1-ARD indicates that ATP binding can stabilize the TRPV-ARD fold.

Figure 5

Figure 5. Effect of ATP binding on protein stability in rTRPV1-ARD. (A) Structure of TRPV1-ARD (gray) bound to ATP (green, sticks), with buried Cys157 highlighted (spheres). (B) TRPV1-ARD is modified at cysteine residues by PEG-maleimide (mPEG), causing an electrophoretic mobility shift on a Coomassie-stained SDS gel. Abbreviations: WT, wild type; CL, a cysteine-less variant; C157, CL-TRPV1-ARD C157 single-cysteine variant. Shown is a representative Coomassie-stained gel from one of three experiments. (C) Time course for modification of single-cysteine TRPV1-ARD variants C157 and C362 with 0.5 mM mPEG at room temperature. (D) Data from four experiments like that depicted in panel C were quantified, and the mean ± standard deviation was plotted. (E and F) Molecular dynamics simulation in which the termini of the ATP-bound TRPV1-ARD (E) or TRPV1-ARD structure with ATP removed prior to equilibrating the system (F) are pulled apart at a rate of 20 nm/ns. Superimposed are the structures at the start (gold) and end (blue) of the simulations. (G and H) Root-mean-square deviation of each Cα atom over the course of the simulation mapped onto the starting models with (G) or without (H) ATP. The change in color from blue to red indicates changes in rmsd from 0 to 80 Å. Simulations in which the termini were pulled apart at a rate of 2 nm/ns gave similar results. See Table S3 of the Supporting Information for experimental details.

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To further confirm our results, we used steered molecular dynamics (SMD) (45) to pull the termini of TRPV1-ARD and reveal stable regions during protein mechanical unfolding in silico. The N- and C-termini were separated at a constant velocity of 2 or 20 nm/ns in the presence or absence of bound ATP. TRPV1-ARD simulated in the absence of bound ATP unfolded at both ends, with repeats 6 and then 1 unraveling during the time periods of independent simulations performed at either stretching speed (Figure 5E,F). In contrast, when ATP, bound to repeats 1–3, was included in the simulations, repeat 6 and much of repeat 5 unravelled, while repeat 1 remained essentially intact (Figure 5G,H). Unfolding forces were similar in the presence or absence of bound ATP at fast stretching speeds, but slightly larger for the ATP-bound ARD in the slower stretching simulations (Figure S2 of the Supporting Information). These simulations therefore support the idea that ATP stabilizes the surrounding local TRPV1-ARD structure. Dynamic ligand-induced changes in TRPV-ARD stability may therefore provide a regulatory mechanism for channel sensitivity and activation.

Structural Analysis of TRPV4 Mutations Associated with Human Diseases

Many mutations in the TRPV4 gene have been associated with inherited diseases. (23, 24) Within the ARD, at least 15 mutations at 12 residues have been reported to cause autosomal dominant diseases classified as either neuropathies or skeletal dysplasias (Figure 6A). (46-48) Mutations at arginine residues located on the convex face of hTRPV4-ARD are associated with neuropathies (Figure 6B). (49) Arg232 and Arg269 are located on Fingers 2 and 3, respectively, while Arg315 and Arg316 are located at the base of Finger 4. In contrast, eight residues whose mutations are associated with skeletal dysplasias (Glu183, Lys197, Leu199, Glu278, Thr295, Ile331, Asp333, and Val342) are spread on both fingers and helices, primarily on the concave face of hTRPV4-ARD (Figure 2B). Therefore, mapping the available genetic data on the hTRPV4-ARD structure suggests that the phenotypic differences of the diseases may be related to the differing functional properties of the concave and convex ARD surfaces.

Figure 6

Figure 6. Mutations associated with human diseases in hTRPV4-ARD. (A) Positions of mutations associated with human inherited diseases that lie within hTRPV4-ARD. Abbreviations: SEDM, spondyloepiphyseal dysplasia, type Maroteaux; SMDK, spondylometaphyseal dysplasia, type Kozolowski; MD, metatropic dysplasia; SMA, spinal muscular atrophy; SPMA, scapuloperoneal spinal muscular atrophy; CMTC2, Charcot-Marie-Tooth disease type 2C; HMSN2C, hereditary motor and sensory neuropathy 2C. This figure was inspired by ref 51. (B) Location of the disease-causing mutations within TRPV4-ARD. Shown as spheres are 12 residue positions at which a total of 15 mutations causing human inherited diseases have been identified. The ATP molecule is shown as sticks. Skeletal dysplasia and neurophathy mutations are depicted as green and blue spheres, respectively. (C) Leu199 is located at the hydrophobic interface between ANK2 and ANK3. (D) Glu183 and Arg232 form a salt bridge on the convex face of TRPV4-ARD.

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To gain insights into possible biochemical mechanisms connecting the mutations with disease phenotypes, we generated mutant hTRPV4-ARD corresponding to 13 of the 15 disease-causing mutations located within the ARD. We were not able to produce recombinant T295A hTRPV4-ARD because of its low level of protein expression and/or stability. The inability of T295A to fold stably when expressed in E. coli and the influence of nucleotide ligands on the thermal stability of hTRPV4-ARD suggest that several mutations could affect protein folding and/or stability. We therefore tested the thermal stability of wild-type hTRPV4-ARD and the 13 purified hTRPV4-ARD mutants by CD spectroscopy. Wild-type hTRPV4-ARD had a melting temperature (Tm) of 37.93 ± 0.08 °C (note that a phosphate-based buffer was used for these experiments). The Tm values of most mutants were significantly different from that of the wild type, except for those of R232C and I331T [38.1 ± 0.1 and 37.97 ± 0.07 °C, respectively (Table S4 and Figure S3 of the Supporting Information)]. Most mutations resulted in a significantly lower Tm values, although R269C showed a significantly higher Tm (38.6 ± 0.2 °C) than the wild type (Table S4 of the Supporting Information). L199F and E183K showed the more severe reductions in Tm (32.9 ± 0.1 and 33.78 ± 0.06 °C, respectively). Leu199 is buried and tightly packed between inner helices 2 and 3, and therefore, its substitution with a larger phenylalanine side chain likely affects the protein thermal stability by disrupting the hydrophobic core packing arrangement (Figure 6C). Glu183 is on the surface of Finger 2 and forms a salt bridge to Arg232 in some of the hTRPV4-ARD structures (Figure 6D). However, simple disruption of this salt bridge is unlikely to cause this decrease in stability because the complementary R232C neutralizing mutation did not significantly alter the Tm of hTRPV4-ARD. The E183K charge reversal may cause electrostatic repulsion between the ANK fingers due to the proximity of Arg232, while the R232C mutation, disrupting the salt bridge, would not actively cause electrostatic repulsion. Overall, the averaged Tm of skeletal dysplasia mutants (35.6 ± 1.6 °C; n = 24) is significantly lower than those of the wild type (37.93 ± 0.08 °C; n = 8; p = 0.0005) and neuropathy mutants (36.9 ± 1.5 °C; n = 15; p = 0.017), although not all mutants obey this trend (Figure 7A).

Figure 7

Figure 7. Thermal stability and ATP binding of hTRPV4-ARD mutants associated with inherited diseases. (A) The Tm determined by CD spectrometry in a phosphate-based buffer is plotted for wild-type and mutant hTRPV4-ARDs. The statistical significance is shown in Table S4 of the Supporting Information. (B) Coomassie-stained gels show wild-type and mutant TRPV4-ARDs loaded (top) and bound to ATP-agarose (bottom). (C) Normalized intensity of recovered protein (mean ± SD; n = 3). The statistical significance of the change in binding to ATP-agarose with respect to wild type (WT) was determined by a multiple-comparison test using Dunnett’s method, with p < 0.05 and p < 0.01 indicated by one asterisk and two asterisks, respectively.

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ATP Binding by hTRPV4-ARD Mutants

Most disease-associated mutations in TRPV4-ARD cause substitutions of surface-exposed side chains. This suggests that interactions with other factors, such as other regions of the TRPV4 protein and/or regulatory ligands, are affected. Because ATP is a known ligand, we tested the ability of the hTRPV4-ARDs with disease-causing mutations to bind ATP using an ATP-agarose pull-down assay (Figure 7B,C). Four mutants, K197R, R232C, R269H, and V342F, showed significantly weakened ATP binding. Lys197, which directly interacts with ATP in our ATP-bound hTRPV4-ARD structure (Figure 3B), showed one of the greatest reductions in the level of ATP binding. This is also consistent with previously published experiments in which corresponding residues Lys183 of cTRPV4-ARD and Lys160 of rTRPV1-ARD were shown to be crucial for ATP binding. (7, 8) In contrast, the reduction in the level of ATP binding observed for R232C and R269H is surprising because both residues are on the surface opposite the ATP-binding site (Figure 6B). Similarly, ATP binding is significantly enhanced by the E183K and E278K mutations, although these residues also have no direct interaction with ATP. These results suggest that, although the interaction of ATP is specific to the ATP-binding site observed in our structure, (8) it is influenced by the general electrostatic properties of hTRPV4-ARD. Because intracellular ATP sensitizes TRPV4 channel activity, (8) enhanced ATP binding may result in higher channel activity, leading to constitutive basal activity. However, although several mutations either impaired or enhanced ATP binding, there was no correlation between the disease phenotypes and the ATP-binding phenotype.
In summary, enhanced ATP binding in E183K and E278K could contribute to the constitutive TRPV4 activity that is thought to lead to the TRPV4-linked disease phenotypes. However, it is difficult to explain the different disease phenotypes observed, including neuropathy and skeletal dysplasia, on the basis of only changes in thermal stability or ATP binding (Figure 7A,C), suggesting complex and unidentified mechanisms for regulating TRPV4 channel activity in different tissues.

Discussion

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In this study, we determined the structures of ATP-free and -bound hTRPV4-ARD and compared them with the structures of other TRPV-ARDs. Structural analyses revealed the flexible Finger 3 as a possible switch for regulating channel activity. Biochemical analyses and MD simulations indicated that ATP contributed to protein stability in TRPV-ARDs possessing ATP binding ability. Finally, a series of biochemical analyses of the disease-associated mutants showed that several have impaired thermal stability and/or ATP binding ability.
The new pair of crystal structures of the ATP-free and -bound forms of TRPV4-ARD revealed minimal conformational changes in the presence of bound ligand. The long and flexible Finger 3 undergoes the largest conformational change. In the process, a patch of aromatic residues is affected, such that a number of these aromatic residues are buried in the presence of ATP. This suggests that a regulatory signal could be exposed or hidden by a Finger 3 conformational change, to affect the sensitization of TRPV4 and the related TRPV1 channel by ATP. (7, 8) In other words, ATP binding may control the accessibility of a regulatory surface, which could interact intramolecularly with other parts of the TRPV4 channel or intermolecularly with additional regulatory factors. One candidate for such a regulatory factor is calmodulin, which was shown to require residues within the ATP-binding site for binding to TRPV4-ARD. (8)
It is interesting that the ATP-bound ARD structures of both TRPV1 and TRPV4, with their packed forms of Finger 3, are similar to those of TRPV2 and TRPV6, which lack the ability to bind ATP. Although it does not bind ATP, in phylogenetic analyses TRPV2 clusters with TRPV1, TRPV3, and TRPV4, which can bind ATP. This clustering and the fact that most ATP-binding residues, including two critical lysines, are conserved in TRPV2 (8) suggest that TRPV2 may have lost its ATP binding ability in a relatively recent evolutionary process. A previous study attempted to generate a TRPV2-ARD mutant that could bind ATP and/or CaM by introducing two mutations: D78N, which neutralizes a negatively charged side chain in the proximity of the phosphate interaction site, and H165Q, which could restore the adenine-binding pocket. (8) Although neither of the single mutants bound ATP, the D78N/H165Q double mutant bound ATP weakly but significantly, suggesting that additional residues are essential for ATP binding. Our structure of ATP-bound TRPV4-ARD suggests a candidate, Phe231, which is conserved in the rTRPV1-ARD ATP-binding site (Tyr194) but not in TRPV2 and TRPV6 (Figure 3D). Comparing the TRPV-ARD structures suggests that this aromatic residue buttresses the adenine-binding pocket.
Several lines of evidence indicate that ATP binding increases the protein stability of both TRPV1- and TRPV4-ARD (Figures 4 and 5 and Figure S1 of the Supporting Information). Interestingly, TRPV4-ARD showed a Tm near body temperature (37.1 ± 0.1 °C), suggesting that the ARD could be quite sensitive to changes in physiological temperature. In such a situation, a small but significant increase in Tm induced by binding of a ligand such as that observed here for ATP (37.9 ± 0.2 °C) may make a large contribution to protein stability at body temperature. It has been reported that TRPV4 is activated by warm temperatures (threshold for activation of ∼34 °C) and shows basal constitutive activity around body temperature. (13) Furthermore, it has been suggested that some unfolding event may be responsible for the thermosensitivity of TRP channels. (50) Therefore, ARD stabilization by ligand binding may fine-tune TRPV4 function and basal activity levels, although the relationship between the stability of ARD and channel activity remains speculative.
TRPV4 is unusual among the TRPV ion channels because a large number of dominant missense mutations that cause a whole spectrum of human skeletal dysplasias and neurodegenerative diseases have been identified. In particular, the neurodegenerative disease-causing mutations all localize to one surface of the ARD. (49) In contrast, the skeletal dysplasia mutations spread throughout the protein, (51) although a number of them are found in the ARD. We studied a total of 15 mutations at 12 positions localized to the ARD, five neurodegenerative disease mutations and 10 skeletal dysplasia mutations. Recently, two new mutations, Q239H (associated with skeletal dysplasia and located within the adenine pocket of the ATP-binding site) and R316H (associated with CMT2C like the previously identified R316C), have been reported in the ARD. (52, 53) Several hTRPV4 mutants, including both neurodegenerative mutations (R232C, R269C, R269H, R315W, and R316C) and skeletal dysplasia mutations (I331F and D333G), cause high basal activity and enhanced response to stimuli when expressed heterologously in HEK293 cells and were inferred to cause cell death because of high Ca2+ influx. (26-28, 46, 53-55) The constitutive activity of hTRPV4 mutants is consistent with their dominant disease phenotypes. However, how different tissue-specific phenotypes are observed when both types of mutations result in a similar cellular phenotype remains unexplained.
We therefore generated 13 of the ARD mutant proteins and compared several of their biochemical properties. We did find several mutations causing significant enhancement or reduction in ARD thermal stability and/or ATP binding. These changes could play a part in the disease mechanisms. Most hTRPV4-ARD mutants showed significantly reduced thermal stability, suggesting that the ARD thermal stability may contribute to the regulation of channel activity. For example, the ARD stability could alter the functional channel population as the N-terminus of TRPV4 has been reported to be important for tetramer assembly. (56) However, a lower Tm does not provide a general correlation to TRPV4 function because R269C, which showed a Tm higher than that of the wild type, has been reported to cause the constitutive activity of the channel. (27, 28) Of note, the enhanced ATP binding by two skeletal dysplasia mutations, E183K and E278K, suggests a mechanism for increased TRPV4 activity and Ca2+ influx because intracellular ATP has been shown to sensitize TRPV4. (8)
Biochemical analyses in this study suggested two possible mechanisms for how mutations in TRPV4-ARD affect channel activity: reduced ARD thermal stability and enhanced ATP binding. However, it is difficult to explain the disease-specific segregation on the basis of only these two biochemical phenotypes, suggesting that additional mechanisms are likely at play in the regulation of the TRPV4 channel. We also did not see a correlation between thermal stability and ATP binding, suggesting that there is no clear functional relationship between these two phenotypes. Our results therefore leave us with two broad hypotheses. (1) Disease-causing TRPV4 mutations affect diverse regulatory mechanisms, converging to bone or neuron-related phenotypes in how they ultimately affect TRPV4 activity. (2) The most important common mechanisms remain to be identified. That is, there could be regulatory factors or mechanisms specific to each tissue. Further understanding of the mechanisms that cause the human inherited diseases will therefore require the identification of tissue-specific biochemical phenotypes, as well as additional cell biology and electrophysiology studies to link biochemical findings to ion channel function.

Supporting Information

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TRPV-ARD structures used in this study (Table S1), structural similarity between TRPV4-ARD and other TRPV-ARDs (Table S2), molecular dynamics simulations of rat TRPV1-ARD (Table S3), Tm values of wild-type and mutant TRPV4-ARD proteins (Table S4), hTRPV4-ARD cysteine modification assay (Figure S1), stabilities of TRPV1-ARD in equilibrium and SMD simulations (Figure S2), and thermal stabilities of wild-type and mutant TRPV4-ARD proteins (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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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

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  • Corresponding Author
    • Rachelle Gaudet - Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, United States;  Email: [email protected]
  • Authors
    • Hitoshi Inada - Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, United States
    • Erik Procko - Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, United States
    • Marcos Sotomayor - Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, United States;  Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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We thank David Neau for assistance with data collection, Dr. Wilhelm A. Weihofen for help with data processing, Dr. Charlotte J. Sumner for providing human TRPV4 cDNA, Dr. Ute Hellmich for comments, and current and former lab members for technical help and discussions.

Abbreviations

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ANK

ankyrin repeat

ARD

ankyrin repeat domain

CaM

calmodulin

CD

circular dichroism

DTT

dithiothreitol

EDTA

ethylenediaminetetraacetic acid

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IPTG

isopropyl β-d-thiogalactopyranoside

MD

molecular dynamics

PAGE

polyacrylamide gel electrophoresis

PDB

Protein Data Bank

PEG

polyethylene glycol

rmsd

root-mean-square deviation

SDS

sodium dodecyl sulfate

SMD

steered molecular dynamics

TRP

transient receptor potential

TRPV

TRP vanilloid.

References

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    Mochizuki, T., Sokabe, T., Araki, I., Fujishita, K., Shibasaki, K., Uchida, K., Naruse, K., Koizumi, S., Takeda, M., and Tominaga, M. (2009) The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures J. Biol. Chem. 284, 21257– 21264
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    There is no corresponding record for this reference.
  19. 19
    Sokabe, T. and Tominaga, M. (2010) The TRPV4 cation channel: A molecule linking skin temperature and barrier function Commun. Integr. Biol. 3, 619– 621
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    19
    The TRPV4 cation channel: A molecule linking skin temperature and barrier function
    Sokabe Takaaki; Tominaga Makoto
    Communicative & integrative biology (2010), 3 (6), 619-21 ISSN:.
    The skin barrier function is indispensable for terrestrial animals to avoid dehydration. The function is achieved by a hydrophobic cornified layer consisting of dead keratinocytes and lipids, and by an intercellular junction barrier formed among differentiated keratinocytes. A recent report demonstrated that TRPV4, one of the temperature-sensitive cation channels, contributes to the formation and maintenance of the intercellular junction-dependent barrier in the skin. TRPV4 associates with the E-cadherin complex via β-catenin, and thereby participates in the promotion of cell-cell junction development. TRPV4 allows influx of Ca(2+) ions from the extracellular space at physiological skin temperatures. The Ca(2+) influx induces Rho activation and promotes actin fiber organization and junction formation, thereby augmenting barrier integrity. Indeed, the intercellular junction structures and the skin barrier function were impaired in TRPV4-deficeint mice. This novel role of TRPV4 in keratinocytes may explain the significant correlation between temperature and the condition of skin.>
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  20. 20
    Shibasaki, K., Suzuki, M., Mizuno, A., and Tominaga, M. (2007) Effects of body temperature on neural activity in the hippocampus: Regulation of resting membrane potentials by transient receptor potential vanilloid 4 J. Neurosci. 27, 1566– 1575
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    20
    Effects of body temperature on neural activity in the hippocampus: regulation of resting membrane potentials by transient receptor potential vanilloid 4
    Shibasaki, Koji; Suzuki, Makoto; Mizuno, Atsuko; Tominaga, Makoto
    Journal of Neuroscience (2007), 27 (7), 1566-1575CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Physiol. body temp. is an important determinant for neural functions, and it is well established that changes in temp. have dynamic influences on hippocampal neural activities. However, the detailed mol. mechanisms have never been clarified. Here, we show that hippocampal neurons express functional transient receptor potential vanilloid 4 (TRPV4), one of the thermosensitive TRP (transient receptor potential) channels, and that TRPV4 is constitutively active at physiol. temp. Activation of TRPV4 at 37°C depolarized the resting membrane potential in hippocampal neurons by allowing cation influx, which was obsd. in wild-type (WT) neurons, but not in TRPV4-deficient (TRPV4KO) cells, although dendritic morphol., synaptic marker clustering, and synaptic currents were indistinguishable between the two genotypes. Furthermore, current injection studies revealed that TRPV4KO neurons required larger depolarization to evoke firing, equiv. to WT neurons, indicating that TRPV4 is a key regulator for hippocampal neural excitabilities. We conclude that TRPV4 is activated by physiol. temp. in hippocampal neurons and thereby controls their excitability.
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  21. 21
    Alessandri-Haber, N., Dina, O. A., Joseph, E. K., Reichling, D., and Levine, J. D. (2006) A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators J. Neurosci. 26, 3864– 3874
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    21
    A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators
    Alessandri-Haber, Nicole; Dina, Olayinka A.; Joseph, Elizabeth K.; Reichling, David; Levine, Jon D.
    Journal of Neuroscience (2006), 26 (14), 3864-3874CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    The transient receptor potential vanilloid 4 (TRPV4) is a primary afferent transducer that plays a crucial role in neuropathic hyperalgesia for osmotic and mech. stimuli, as well as in inflammatory mediator-induced hyperalgesia for osmotic stimuli. In view of the clin. importance of mech. hyperalgesia in inflammatory states, the present study investigated the role of TRPV4 in mech. hyperalgesia induced by inflammatory mediators and the second-messenger pathways involved. Intradermal injection of either the inflammogen carrageenan or a soup of inflammatory mediators enhanced the nocifensive paw-withdrawal reflex elicited by hypotonic or mech. stimuli in rat. Spinal administration of TRPV4 antisense oligodeoxynucleotide blocked the enhancement without altering baseline nociceptive threshold. Similarly, in TRPV4-/- knock-out mice, inflammatory soup failed to induce any significant mech. or osmotic hyperalgesia. In vitro investigation showed that inflammatory mediators engage the TRPV4-mediated mechanism of sensitization by direct action on dissocd. primary afferent neurons. Addnl. behavioral observations suggested that multiple mediators are necessary to achieve sufficient activation of the cAMP pathway to engage the TRPV4-dependent mechanism of hyperalgesia. In addn., direct activation of protein kinase A or protein kinase C ε, two pathways that mediate inflammation-induced mech. hyperalgesia, also induced hyperalgesia for both hypotonic and mech. stimuli that was decreased by TRPV4 antisense and absent in TRPV4-/- mice. We conclude that TRPV4 plays a crucial role in the mech. hyperalgesia that is generated by the concerted action of inflammatory mediators present in inflamed tissues.
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  22. 22
    Masuyama, R., Vriens, J., Voets, T., Karashima, Y., Owsianik, G., Vennekens, R., Lieben, L., Torrekens, S., Moermans, K., Vanden Bosch, A., Bouillon, R., Nilius, B., and Carmeliet, G. (2008) TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts Cell Metab. 8, 257– 265
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    22
    TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts
    Masuyama, Ritsuko; Vriens, Joris; Voets, Thomas; Karashima, Yuji; Owsianik, Grzegorz; Vennekens, Rudi; Lieben, Liesbet; Torrekens, Sophie; Moermans, Karen; Vanden Bosch, An; Bouillon, Roger; Nilius, Bernd; Carmeliet, Geert
    Cell Metabolism (2008), 8 (3), 257-265CODEN: CMEEB5; ISSN:1550-4131. (Cell Press)
    Calcium signaling controls multiple cellular functions and is regulated by the release from internal stores and entry from extracellular fluid. In bone, osteoclast differentiation is induced by RANKL (receptor activator of NF-κB ligand)-evoked intracellular Ca2+ oscillations, which trigger nuclear factor-activated T cells (NFAT) c1-responsive gene transcription. However, the Ca2+ channels involved remain largely unidentified. Here we show that genetic ablation in mice of Trpv4, a Ca2+-permeable channel of the transient receptor potential (TRP) family, increases bone mass by impairing bone resorption. TRPV4 mediates basolateral Ca2+ influx specifically in large osteoclasts when Ca2+ oscillations decline. TRPV4-mediated Ca2+ influx hereby secures intracellular Ca2+ concns., ensures NFATc1-regulated gene transcription, and regulates the terminal differentiation and activity of osteoclasts. In conclusion, our data indicate that Ca2+ oscillations and TRPV4-mediated Ca2+ influx are sequentially required to sustain NFATc1-dependent gene expression throughout osteoclast differentiation, and we propose TRPV4 as a therapeutic target for bone diseases.
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  23. 23
    Nilius, B. and Owsianik, G. (2010) Transient receptor potential channelopathies Pfluegers Arch. 460, 437– 450
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    23
    Transient receptor potential channelopathies
    Nilius, Bernd; Owsianik, Grzegorz
    Pfluegers Archiv (2010), 460 (2), 437-450CODEN: PFLABK; ISSN:0031-6768. (Springer GmbH)
    A review. In the past years, several hereditary diseases caused by defects in transient receptor potential channels (TRP) genes have been described. This review summarizes our current knowledge about TRP channelopathies and their possible pathomechanisms. Based on available genetic indications, we will also describe several putative pathol. conditions in which (mal)function of TRP channels could be anticipated.
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  24. 24
    Verma, P., Kumar, A., and Goswami, C. (2010) TRPV4-mediated channelopathies Channels 4, 319– 328
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    There is no corresponding record for this reference.
  25. 25
    Rock, M. J., Prenen, J., Funari, V. A., Funari, T. L., Merriman, B., Nelson, S. F., Lachman, R. S., Wilcox, W. R., Reyno, S., Quadrelli, R., Vaglio, A., Owsianik, G., Janssens, A., Voets, T., Ikegawa, S., Nagai, T., Rimoin, D. L., Nilius, B., and Cohn, D. H. (2008) Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia Nat. Genet. 40, 999– 1003
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    25
    Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia
    Rock, Matthew J.; Prenen, Jean; Funari, Vincent A.; Funari, Tara L.; Merriman, Barry; Nelson, Stanley F.; Lachman, Ralph S.; Wilcox, William R.; Reyno, Soraya; Quadrelli, Roberto; Vaglio, Alicia; Owsianik, Grzegorz; Janssens, Annelies; Voets, Thomas; Ikegawa, Shiro; Nagai, Toshiro; Rimoin, David L.; Nilius, Bernd; Cohn, Daniel H.
    Nature Genetics (2008), 40 (8), 999-1003CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
    Daniel Cohn and colleagues identify mutations in the gene encoding the calcium-permeable cation channel TRPV4 in families with autosomal dominant brachyolmia. Functional studies show that the mutations result in gain-of-function of channel activation. Daniel Cohn and colleagues identify mutations in the gene encoding the calcium-permeable cation channel TRPV4 in families with autosomal dominant brachyolmia. Functional studies show that the mutations result in gain-of-function of channel activation. The brachyolmias constitute a clin. and genetically heterogeneous group of skeletal dysplasias characterized by a short trunk, scoliosis and mild short stature. Here, we identify a locus for an autosomal dominant form of brachyolmia on chromosome 12q24.1-12q24.2. Among the genes in the genetic interval, we selected TRPV4, which encodes a calcium permeable cation channel of the transient receptor potential (TRP) vanilloid family, as a candidate gene because of its cartilage-selective gene expression pattern. In two families with the phenotype, we identified point mutations in TRPV4 that encoded R616Q and V620I substitutions, resp. Patch clamp studies of transfected HEK cells showed that both mutations resulted in a dramatic gain of function characterized by increased constitutive activity and elevated channel activation by either mechano-stimulation or agonist stimulation by arachidonic acid or the TRPV4-specific agonist 4α-phorbol 12,13-didecanoate (4αPDD). This study thus defines a previously unknown mechanism, activation of a calcium-permeable TRP ion channel, in skeletal dysplasia pathogenesis.
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  26. 26
    Krakow, D., Vriens, J., Camacho, N., Luong, P., Deixler, H., Funari, T. L., Bacino, C. A., Irons, M. B., Holm, I. A., Sadler, L., Okenfuss, E. B., Janssens, A., Voets, T., Rimoin, D. L., Lachman, R. S., Nilius, B., and Cohn, D. H. (2009) Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia Am. J. Hum. Genet. 84, 307– 315
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    26
    Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia
    Krakow, Deborah; Vriens, Joris; Camacho, Natalia; Luong, Phi; Deixler, Hannah; Funari, Tara L.; Bacino, Carlos A.; Irons, Mira B.; Holm, Ingrid A.; Sadler, Laurie; Okenfuss, Ericka B.; Janssens, Annelies; Voets, Thomas; Rimoin, David L.; Lachman, Ralph S.; Nilius, Bernd; Cohn, Daniel H.
    American Journal of Human Genetics (2009), 84 (3), 307-315CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    The spondylometaphyseal dysplasias (SMDs) are a group of short-stature disorders distinguished by abnormalities in the vertebrae and the metaphyses of the tubular bones. SMD Kozlowski type (SMDK) is a well-defined autosomal-dominant SMD characterized by significant scoliosis and mild metaphyseal abnormalities in the pelvis. The vertebrae exhibit platyspondyly and overfaced pedicles similar to autosomal-dominant brachyolmia, which can result from heterozygosity for activating mutations in the gene encoding TRPV4, a calcium-permeable ion channel. Mutation anal. in six out of six patients with SMDK demonstrated heterozygosity for missense mutations in TRPV4, and one mutation, predicting a R594H substitution, was recurrent in four patients. Similar to autosomal-dominant brachyolmia, the mutations altered basal calcium channel activity in vitro. Metatropic dysplasia is another SMD that has been proposed to have both clin. and genetic heterogeneity. Patients with the nonlethal form of metatropic dysplasia present with a progressive scoliosis, widespread metaphyseal involvement of the appendicular skeleton, and carpal ossification delay. Because of some similar radiog. features between SMDK and metatropic dysplasia, TRPV4 was tested as a disease gene for nonlethal metatropic dysplasia. In two sporadic cases, heterozygosity for de novo missense mutations in TRPV4 was found. The findings demonstrate that mutations in TRPV4 produce a phenotypic spectrum of skeletal dysplasias from the mild autosomal-dominant brachyolmia to SMDK to autosomal-dominant metatropic dysplasia, suggesting that these disorders should be grouped into a new bone dysplasia family.
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  27. 27
    Deng, H. X., Klein, C. J., Yan, J., Shi, Y., Wu, Y., Fecto, F., Yau, H. J., Yang, Y., Zhai, H., Siddique, N., Hedley-Whyte, E. T., Delong, R., Martina, M., Dyck, P. J., and Siddique, T. (2010) Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4 Nat. Genet. 42, 165– 169
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    27
    Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4
    Deng, Han-Xiang; Klein, Christopher J.; Yan, Jian-Hua; Shi, Yong; Wu, Yan-Hong; Fecto, Faisal; Yau, Hau-Jie; Yang, Yi; Zhai, Hong; Siddique, Nailah; Hedley-Whyte, E. Tessa; De Long, Robert; Martina, Marco; Dyck, Peter J.; Siddique, Teepu
    Nature Genetics (2010), 42 (2), 165-169CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
    Scapuloperoneal spinal muscular atrophy (SPSMA) and hereditary motor and sensory neuropathy type IIC (HMSN IIC, also known as HMSN2C or Charcot-Marie-Tooth disease type 2C (CMT2C)) are phenotypically heterogeneous disorders involving topog. distinct nerves and muscles. We originally described a large New England family of French-Canadian origin with SPSMA and an American family of English and Scottish descent with CMT2C. We mapped SPSMA and CMT2C risk loci to 12q24.1-q24.31 with an overlapping region between the two diseases. Further anal. reduced the CMT2C risk locus to a 4-Mb region. Here we report that SPSMA and CMT2C are allelic disorders caused by mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 4 (TRPV4). Functional anal. revealed that increased calcium channel activity is a distinct property of both SPSMA- and CMT2C-causing mutant proteins. Our findings link mutations in TRPV4 to altered calcium homeostasis and peripheral neuropathies, implying a pathogenic mechanism and possible options for therapy for these disorders.
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  28. 28
    Landoure, G., Zdebik, A. A., Martinez, T. L., Burnett, B. G., Stanescu, H. C., Inada, H., Shi, Y., Taye, A. A., Kong, L., Munns, C. H., Choo, S. S., Phelps, C. B., Paudel, R., Houlden, H., Ludlow, C. L., Caterina, M. J., Gaudet, R., Kleta, R., Fischbeck, K. H., and Sumner, C. J. (2010) Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C Nat. Genet. 42, 170– 174
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    28
    Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C
    Landoure, Guida; Zdebik, Anselm A.; Martinez, Tara L.; Burnett, Barrington G.; Stanescu, Horia C.; Inada, Hitoshi; Shi, Yi-Jun; Taye, Addis A.; Kong, Ling-Ling; Munns, Clare H.; Choo, Shelly S.; Phelps, Christopher B.; Paudel, Reema; Houlden, Henry; Ludlow, Christy L.; Caterina, Michael J.; Gaudet, Rachelle; Kleta, Robert; Fischbeck, Kenneth H.; Sumner, Charlotte J.
    Nature Genetics (2010), 42 (2), 170-174CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
    Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously assocd. with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-assocd. mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.
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  29. 29
    Jin, X., Touhey, J., and Gaudet, R. (2006) Structure of the N-terminal ankyrin repeat domain of the TRPV2 ion channel J. Biol. Chem. 281, 25006– 25010
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    There is no corresponding record for this reference.
  30. 30
    Otwinowski, Z. and Minor, W. (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode Methods Enzymol. 276, 307– 326
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    30
    Processing of x-ray diffraction data collected in oscillation mode
    Otwinowski, Zbyszek; Minor, Wladek
    Methods in Enzymology (1997), 276 (Macromolecular Crystallography, Part A), 307-326CODEN: MENZAU; ISSN:0076-6879. (Academic)
    Macromol. crystallog. is an iterative process. Rarely do the first crystals provide all the necessary data to solve the biol. problem being studied. Each step benefits from experience learned in previous steps. To monitor the progress, the HKL package provides 2 tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), are provided, and the Bayesian reasoning and multicomponent error model facilitates obtaining the proper error ests. and (2) visualization of the process plays a double role by helping the operator to confirm that the process of data redn., including the resulting statistics, is correct, and allowing one to evaluate problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and redn. has been reached. At that point, the effort should be directed to solving the structure. The methods presented here have been applied to solve a large variety of problems, from inorg. mols. with 5 Å unit cell to rotavirus of 700 Å diam. crystd. in 700 × 1000 × 1400 Å cell. Overall quality of the method was tested by many researchers by successful application of the programs to MAD structure detns.
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  31. 31
    Vagin, A. and Teplyakov, A. (2000) An approach to multi-copy search in molecular replacement Acta Crystallogr. D56, 1622– 1624
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    McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software J. Appl. Crystallogr. 40, 658– 674
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    32
    Phaser crystallographic software
    McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.
    Journal of Applied Crystallography (2007), 40 (4), 658-674CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
    Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
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    Emsley, P. and Cowtan, K. (2004) Coot: Model-building tools for molecular graphics Acta Crystallogr. D60, 2126– 2132
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    33
    Coot: model-building tools for molecular graphics
    Emsley, Paul; Cowtan, Kevin
    Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
    CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
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    Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr. D53, 240– 255
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    Refinement of macromolecular structures by the maximum-likelihood method
    Murshudov, Garib N.; Vagin, Alexei A.; Dodson, Eleanor J.
    Acta Crystallographica, Section D: Biological Crystallography (1997), D53 (3), 240-255CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard)
    A review with many refs. on the math. basis of max. likelihood. The likelihood function for macromol. structures is extended to include prior phase information and exptl. std. uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estg. σA using "free" reflections is described and its effects analyzed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the max.-likelihood residual are consistently better than those obtained from least-squares refinement.
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    Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: Visual molecular dynamics J. Mol. Graphics 14, 27– 38
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    Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable molecular dynamics with NAM J. Comput. Chem. 26, 1781– 1802
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    MacKerell, A. D., Jr., Bashford, D., Bellott, M., Dunbrack, R. L., Jr., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., III, Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiórkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins J. Phys. Chem. B 102, 3586– 3616
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    37
    All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins
    MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E., III; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M.
    Journal of Physical Chemistry B (1998), 102 (18), 3586-3616CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
    New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used exptl. gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the at. charges, were detd. by fitting ab initio interaction energies and geometries of complexes between water and model compds. that represented the backbone and the various side chains. In addn., dipole moments, exptl. heats and free energies of vaporization, solvation and sublimation, mol. vols., and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in a crystal. A detailed anal. of the relationship between the alanine dipeptide potential energy surface and calcd. protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in soln. and in crystals. Extensive comparisons between mol. dynamics simulation and exptl. data for polypeptides and proteins were performed for both structural and dynamic properties. Calcd. data from energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with exptl. crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of mols. of biol. interest.
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  38. 38
    Mackerell, A. D., Jr., Feig, M., and Brooks, C. L., III (2004) Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations J. Comput. Chem. 25, 1400– 1415
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    38
    Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations
    MacKerell, Alexander D., Jr.; Feig, Michael; Brooks, Charles L., III
    Journal of Computational Chemistry (2004), 25 (11), 1400-1415CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    Computational studies of proteins based on empirical force fields represent a powerful tool to obtain structure-function relationships at an at. level, and are central in current efforts to solve the protein folding problem. The results from studies applying these tools are, however, dependent on the quality of the force fields used. In particular, accurate treatment of the peptide backbone is crucial to achieve representative conformational distributions in simulation studies. To improve the treatment of the peptide backbone, quantum mech. (QM) and mol. mech. (MM) calcns. were undertaken on the alanine, glycine, and proline dipeptides, and the results from these calcns. were combined with mol. dynamics (MD) simulations of proteins in crystal and aq. environments. QM potential energy maps of the alanine and glycine dipeptides at the LMP2/cc-pVxZ/MP2/6-31G* levels, where x = D, T, and Q, were detd., and are compared to available QM studies on these mols. The LMP2/cc pVQZ//MP2/6-31G* energy surfaces for all three dipeptides were then used to improve the MM treatment of the dipeptides. These improvements included addnl. parameter optimization via Monte Carlo simulated annealing and extension of the potential energy function to contain peptide backbone .vphi., ψ dihedral crossterms or a .vphi., ψ grid-based energy correction term. Simultaneously, MD simulations of up to seven proteins in their cryst. environments were used to validate the force field enhancements. Comparison with QM and crystallog. data showed that an addnl. optimization of the .vphi., ψ dihedral parameters along with the grid-based energy correction were required to yield significant improvements over the CHARMM22 force field. However, systematic deviations in the treatment of .vphi. and ψ in the helical and sheet regions were evident. Accordingly, empirical adjustments were made to the grid-based energy correction for alanine and glycine to account for these systematic differences. These adjustments lead to greater deviations from QM data for the two dipeptides but also yielded improved agreement with exptl. crystallog. data. These improvements enhance the quality of the CHARMM force field in treating proteins. This extension of the potential energy function is anticipated to facilitate improved treatment of biol. macromols. via MM approaches in general.
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  39. 39
    McCleverty, C. J., Koesema, E., Patapoutian, A., Lesley, S. A., and Kreusch, A. (2006) Crystal structure of the human TRPV2 channel ankyrin repeat domain Protein Sci. 15, 2201– 2206
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    39
    Crystal structure of the human TRPV2 channel ankyrin repeat domain
    McCleverty, Clare J.; Koesema, Eric; Patapoutian, Ardem; Lesley, Scott A.; Kreusch, Andreas
    Protein Science (2006), 15 (9), 2201-2206CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
    TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain 3-4 ankyrin repeats. Here, the authors report the 1st structure from the TRPV channel subfamily, a 1.7-Å resoln. crystal structure of human TRPV2 ARD. The crystal structure revealed a 6-ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contained extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last 2 repeats. The TRPV2 ARD provided the 1st structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.
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  40. 40
    Phelps, C. B., Huang, R. J., Lishko, P. V., Wang, R. R., and Gaudet, R. (2008) Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels Biochemistry 47, 2476– 2484
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    Croy, C. H., Bergqvist, S., Huxford, T., Ghosh, G., and Komives, E. A. (2004) Biophysical characterization of the free IκBα ankyrin repeat domain in solution Protein Sci. 13, 1767– 1777
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    41
    Biophysical characterization of the free IκBα ankyrin repeat domain in solution
    Croy, Carrie Hughes; Bergqvist, Simon; Huxford, Tom; Ghosh, Gourisankar; Komives, Elizabeth A.
    Protein Science (2004), 13 (7), 1767-1777CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
    The crystal structure of IκBα in complex with the transcription factor, nuclear factor κ-B (NF-κB) shows six ankyrin repeats, which are all ordered. Electron d. was not obsd. for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IκBα (67-317) when it is not in complex with NF-κB, we have carried out CD spectroscopy, 8-anilino-1-naphthalenesulfonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange expts. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amt. of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange expts. show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. IκBα unfolds with a Tm of 42°, and forms a sol. aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biol. function of IκBα are discussed.
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  42. 42
    Truhlar, S. M., Torpey, J. W., and Komives, E. A. (2006) Regions of IκBα that are critical for its inhibition of NF-κB·DNA interaction fold upon binding to NF-κB Proc. Natl. Acad. Sci. U.S.A. 103, 18951– 18956
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    Barrick, D., Ferreiro, D. U., and Komives, E. A. (2008) Folding landscapes of ankyrin repeat proteins: Experiments meet theory Curr. Opin. Struct. Biol. 18, 27– 34
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    43
    Folding landscapes of ankyrin repeat proteins: Experiments meet theory
    Barrick, Doug; Ferreiro, Diego U.; Komives, Elizabeth A.
    Current Opinion in Structural Biology (2008), 18 (1), 27-34CODEN: COSBEF; ISSN:0959-440X. (Elsevier B.V.)
    A review. Nearly 6% of eukaryotic protein sequences contain ankyrin repeat (AR) domains, which consist of several repeats and often function in binding. AR proteins show highly cooperative folding despite a lack of long-range contacts. Both theory and expt. converge to explain that formation of the interface between elements is more favorable than formation of any individual repeat unit. IκBα and Notch both undergo partial folding upon binding perhaps influencing the binding free energy. The simple architecture, combined with identification of consensus residues that are important for stability, has enabled systematic perturbation of the energy landscape by single point mutations that affect stability or by addn. of consensus repeats. The folding energy landscapes appear highly plastic, with small perturbations re-routing folding pathways.
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  44. 44
    Salazar, H., Llorente, I., Jara-Oseguera, A., Garcia-Villegas, R., Munari, M., Gordon, S. E., Islas, L. D., and Rosenbaum, T. (2008) A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic Nat. Neurosci. 11, 255– 261
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    44
    A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic
    Salazar, Hector; Llorente, Itzel; Jara-Oseguera, Andres; Garcia-Villegas, Refugio; Munari, Mika; Gordon, Sharona E.; Islas, Leon D.; Rosenbaum, Tamara
    Nature Neuroscience (2008), 11 (3), 255-261CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
    Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compds. found in garlic, onion, mustard and cinnamon exts., activation of TRPV1 by these exts. remains controversial. Here the authors establish that TRPV1 is activated by pungent exts. from onion and garlic, as well as by allicin, the active compd. in these prepns., and participates together with TRPA1 in the pain-related behavior induced by this compd. The authors found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects the authors obsd. The authors findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the mol. basis of noxious stimuli detection.
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  45. 45
    Isralewitz, B., Gao, M., and Schulten, K. (2001) Steered molecular dynamics and mechanical functions of proteins Curr. Opin. Struct. Biol. 11, 224– 230
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    45
    Steered molecular dynamics and mechanical functions of proteins
    Isralewitz, Barry; Gao, Mu; Schulten, Klaus
    Current Opinion in Structural Biology (2001), 11 (2), 224-230CODEN: COSBEF; ISSN:0959-440X. (Elsevier Science Ltd.)
    A review with 50 refs. At. force microscopy of single mols., steered mol. dynamics and the theory of stochastic processes have established a new field that investigates mech. functions of proteins, such as ligand-receptor binding/unbinding and elasticity of muscle proteins during stretching. The combination of these methods yields information on the energy landscape that controls mech. function and on the force-bearing components of proteins, as well as on the underlying phys. mechanisms.
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  46. 46
    Camacho, N., Krakow, D., Johnykutty, S., Katzman, P. J., Pepkowitz, S., Vriens, J., Nilius, B., Boyce, B. F., and Cohn, D. H. (2010) Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia Am. J. Med. Genet., Part A 152A, 1169– 1177
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    46
    Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia
    Camacho, Natalia; Krakow, Deborah; Johnykutty, Sharlin; Katzman, Philip J.; Pepkowitz, Samuel; Vriens, Joris; Nilius, Bernd; Boyce, Brendan F.; Cohn, Daniel H.
    American Journal of Medical Genetics, Part A (2010), 152A (5), 1169-1177CODEN: AJMGB8; ISSN:1552-4825. (Wiley-Liss, Inc.)
    Metatropic dysplasia is a clin. heterogeneous skeletal dysplasia characterized by short extremities, a short trunk with progressive kyphoscoliosis, and craniofacial abnormalities that include a prominent forehead, midface hypoplasia, and a squared-off jaw. Dominant mutations in the gene encoding TRPV4, a calcium permeable ion channel, were identified all 10 of a series of metatropic dysplasia cases, ranging in severity from mild to perinatal lethal. These data demonstrate that the lethal form of the disorder is dominantly inherited and suggest locus homogeneity in the disease. Electrophysiol. studies demonstrated that the mutations activate the channel, indicating that the mechanism of disease may result from increased calcium in chondrocytes. Histol. studies in two cases of lethal metatropic dysplasia revealed markedly disrupted endochondral ossification, with reduced nos. of hypertrophic chondrocytes and presence of islands of cartilage within the zone of primary mineralization. These data suggest that altered chondrocyte differentiation in the growth plate leads to the clin. findings in metatropic dysplasia.
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  47. 47
    Dai, J., Kim, O. H., Cho, T. J., Schmidt-Rimpler, M., Tonoki, H., Takikawa, K., Haga, N., Miyoshi, K., Kitoh, H., Yoo, W. J., Choi, I. H., Song, H. R., Jin, D. K., Kim, H. T., Kamasaki, H., Bianchi, P., Grigelioniene, G., Nampoothiri, S., Minagawa, M., Miyagawa, S. I., Fukao, T., Marcelis, C., Jansweijer, M. C., Hennekam, R. C., Bedeschi, F., Mustonen, A., Jiang, Q., Ohashi, H., Furuichi, T., Unger, S., Zabel, B., Lausch, E., Superti-Furga, A., Nishimura, G., and Ikegawa, S. (2010) Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family J. Med. Genet. 47, 704– 709
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    47
    Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family
    Dai, J.; Kim, O.-H.; Cho, T.-J.; Schmidt-Rimpler, M.; Tonoki, H.; Takikawa, K.; Haga, N.; Miyoshi, K.; Kitoh, H.; Yoo, W.-J.; Choi, I.-H.; Song, H.-R.; Jin, D.-K.; Kim, H.-T.; Kamasaki, H.; Bianchi, P.; Grigelioniene, G.; Nampoothiri, S.; Minagawa, M.; Miyagawa, S.-i.; Fukao, T.; Marcelis, C.; Jansweijer, M. C. E.; Hennekam, R. C. M.; Bedeschi, F.; Mustonen, A.; Jiang, Q.; Ohashi, H.; Furuichi, T.; Unger, S.; Zabel, B.; Lausch, E.; Superti-Furga, A.; Nishimura, G.; Ikegawa, S.
    Journal of Medical Genetics (2010), 47 (10), 704-709CODEN: JMDGAE; ISSN:0022-2593. (BMJ Publishing Group)
    Mutations in TRPV4, a gene that encodes a Ca2+ permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. To examine TRPV4 mutation spectrum and phenotype-genotype assocn., we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471 del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An assocn. between the position of mutations and the disease phenotype was also obsd. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been obsd. at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic labs. establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.
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  48. 48
    Nishimura, G., Dai, J., Lausch, E., Unger, S., Megarbane, A., Kitoh, H., Kim, O. H., Cho, T. J., Bedeschi, F., Benedicenti, F., Mendoza-Londono, R., Silengo, M., Schmidt-Rimpler, M., Spranger, J., Zabel, B., Ikegawa, S., and Superti-Furga, A. (2010) Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2), and parastremmatic dysplasia are caused by TRPV4 mutations Am. J. Med. Genet., Part A 152A, 1443– 1449
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    Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2), and parastremmatic dysplasia are caused by TRPV4 mutations
    Nishimura Gen; Dai Jin; Lausch Ekkehart; Unger Sheila; Megarbane Andre; Kitoh Hiroshi; Kim Ok Hwa; Cho Tae-Joon; Bedeschi Francesca; Benedicenti Francesco; Mendoza-Londono Roberto; Silengo Margherita; Schmidt-Rimpler Maren; Spranger Jurgen; Zabel Bernhard; Ikegawa Shiro; Superti-Furga Andrea
    American journal of medical genetics. Part A (2010), 152A (6), 1443-9 ISSN:.
    Recent discoveries have established the existence of a family of skeletal dysplasias caused by dominant mutations in TRPV4. This family comprises, in order of increasing severity, dominant brachyolmia, spondylo-metaphyseal dysplasia Kozlowski type, and metatropic dysplasia. We tested the hypothesis that a further condition, Spondylo-epiphyseal dysplasia (SED), Maroteaux type (MIM 184095; also known as pseudo-Morquio syndrome type 2), could be caused by TRPV4 mutations. We analyzed six individuals with Maroteaux type SED, including three who had previously been reported. All six patients were found to have heterozygous TRPV4 mutations; three patients had unreported mutations, while three patients had mutations previously described in association with metatropic dysplasia. In addition, we tested one individual with a distinct rare disorder, parastremmatic dysplasia (MIM 168400). This patient had a common, recurrent mutation seen in several patients with Kozlowski type spondylo-metaphyseal dysplasia. We conclude that SED Maroteaux type and parastremmatic dysplasia are part of the TRPV4 dysplasia family and that TRPV4 mutations show considerable variability in phenotypic expression resulting in distinct clinical-radiographic phenotypes.
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  49. 49
    Zimon, M., Baets, J., Auer-Grumbach, M., Berciano, J., Garcia, A., Lopez-Laso, E., Merlini, L., Hilton-Jones, D., McEntagart, M., Crosby, A. H., Barisic, N., Boltshauser, E., Shaw, C. E., Landoure, G., Ludlow, C. L., Gaudet, R., Houlden, H., Reilly, M. M., Fischbeck, K. H., Sumner, C. J., Timmerman, V., Jordanova, A., and Jonghe, P. D. (2010) Dominant mutations in the cation channel gene transient receptor potential vanilloid 4 cause an unusual spectrum of neuropathies Brain 133, 1798– 1809
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    There is no corresponding record for this reference.
  50. 50
    Clapham, D. E. and Miller, C. (2011) A thermodynamic framework for understanding temperature sensing by transient receptor potential (TRP) channels Proc. Natl. Acad. Sci. U.S.A. 108, 19492– 19497
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    There is no corresponding record for this reference.
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    Dai, J., Cho, T. J., Unger, S., Lausch, E., Nishimura, G., Kim, O. H., Superti-Furga, A., and Ikegawa, S. (2010) TRPV4-pathy, a novel channelopathy affecting diverse systems J. Hum. Genet. 55, 400– 402
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    TRPV4-pathy, a novel channelopathy affecting diverse systems
    Dai Jin; Cho Tae-Joon; Unger Sheila; Lausch Ekkehart; Nishimura Gen; Kim Ok-Hwa; Superti-Furga Andrea; Ikegawa Shiro
    Journal of human genetics (2010), 55 (7), 400-2 ISSN:.
    Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a calcium-permeable nonselective cation channel of unknown biological function. TRPV4 mutation was first identified in brachyolmia, and then in a spectrum of autosomal-dominant skeletal dysplasias, which includes Kozlowski type of spondylometaphyseal dysplasia, metatropic dysplasia, Maroteaux type of spondyloepiphyseal dysplasia and parastremmatic dysplasia. Recently, TRPV4 mutation has also been identified in a spectrum of neuromuscular diseases that includes congenital distal spinal muscular atrophy (SMA), scapuloperoneal SMA, and hereditary motor and sensory neuropathy type IIC. These diverse spectrums of diseases compose a novel channelopathy, TRPV4-pathy, which could further include polygenic traits such as serum sodium concentration and a chronic obstructive pulmonary disease. In this review, we clarified the TRPV4 mutation spectrum, and discussed the phenotypic complexity of TRPV4-pathy and its pathogenic mechanisms. TRPV4-pathy may extend further to other monogenic and polygenic diseases.
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  52. 52
    Andreucci, E., Aftimos, S., Alcausin, M., Haan, E., Hunter, W., Kannu, P., Kerr, B., McGillivray, G., Gardner, R. M., Patricelli, M. G., Sillence, D., Thompson, E., Zacharin, M., Zankl, A., Lamande, S. R., and Savarirayan, R. (2011) TRPV4 related skeletal dysplasias: A phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families Orphanet. J. Rare Dis. 6, 37
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    TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families
    Andreucci Elena; Aftimos Salim; Alcausin Melanie; Haan Eric; Hunter Warwick; Kannu Peter; Kerr Bronwyn; McGillivray George; McKinlay Gardner R J; Patricelli Maria G; Sillence David; Thompson Elizabeth; Zacharin Margaret; Zankl Andreas; Lamande Shireen R; Savarirayan Ravi
    Orphanet journal of rare diseases (2011), 6 (), 37 ISSN:.
    BACKGROUND: The TRPV4 gene encodes a calcium-permeable ion-channel that is widely expressed, responds to many different stimuli and participates in an extraordinarily wide range of physiologic processes. Autosomal dominant brachyolmia, spondylometaphyseal dysplasia Kozlowski type (SMDK) and metatropic dysplasia (MD) are currently considered three distinct skeletal dysplasias with some shared clinical features, including short stature, platyspondyly, and progressive scoliosis. Recently, TRPV4 mutations have been found in patients diagnosed with these skeletal phenotypes. METHODS AND RESULTS: We critically analysed the clinical and radiographic data on 26 subjects from 21 families, all of whom had a clinical diagnosis of one of the conditions described above: 15 with MD; 9 with SMDK; and 2 with brachyolmia. We sequenced TRPV4 and identified 9 different mutations in 22 patients, 4 previously described, and 5 novel. There were 4 mutation-negative cases: one with MD and one with SMDK, both displaying atypical clinical and radiographic features for these diagnoses; and two with brachyolmia, who had isolated spine changes and no metaphyseal involvement. CONCLUSIONS: Our data suggest the TRPV4 skeletal dysplasias represent a continuum of severity with areas of phenotypic overlap, even within the same family. We propose that AD brachyolmia lies at the mildest end of this spectrum and, since all cases described with this diagnosis and TRPV4 mutations display metaphyseal changes, we suggest that it is not a distinct entity but represents the mildest phenotypic expression of SMDK.
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  53. 53
    Klein, C. J., Shi, Y., Fecto, F., Donaghy, M., Nicholson, G., McEntagart, M. E., Crosby, A. H., Wu, Y., Lou, H., McEvoy, K. M., Siddique, T., Deng, H. X., and Dyck, P. J. (2011) TRPV4 mutations and cytotoxic hypercalcemia in axonal Charcot-Marie-Tooth neuropathies Neurology 76, 887– 894
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    There is no corresponding record for this reference.
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    Fecto, F., Shi, Y., Huda, R., Martina, M., Siddique, T., and Deng, H. X. (2011) Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies J. Biol. Chem. 286, 17281– 17291
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    Loukin, S., Su, Z., and Kung, C. (2011) Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations PLoS One 6, e19533
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    There is no corresponding record for this reference.
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    Schaefer, M. (2005) Homo- and heteromeric assembly of TRP channel subunits Pfluegers Arch. 451, 35– 42
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    56
    Homo- and heteromeric assembly of TRP channel subunits
    Schaefer, Michael
    Pfluegers Archiv (2005), 451 (1), 35-42CODEN: PFLABK; ISSN:0031-6768. (Springer GmbH)
    A review. Mammalian homologs of the Drosophila melanogaster transient receptor potential (TRP) channels are the 2nd largest cation channel family within the superfamily of hexahelical cation channels. Most mammalian TRP channels function as homo-oligomers and mediate monovalent or divalent cation entry upon activation by a variety of stimuli. Because native TRP channels may be multimeric proteins of possibly complex compn., it has been difficult to compare cation conductances in native tissues to those of clearly defined homomeric TRP channel complexes in living cells. Therefore, the possibility of heteromeric TRP channel assembly has been investigated in recent years by several groups. As a major conclusion of these studies, most heteromeric TRP channel complexes appear to consist of subunit combinations only within relatively narrow confines of phylogenetic subfamilies. Although the general capability of heteromer formation between closely related TRP channel subunits is now clearly established, investigators are only beginning to understand whether these heteromeric complexes are of physiol. significance. Here, the author summarizes current knowledge on the promiscuity and specificity of the assembly of channel complexes composed of TRPC-, TRPV-, and TRPM-subunits of mammalian TRP channels.
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    Figure 1

    Figure 1. Structural comparison of human and chicken TRPV4-ARDs. (A) Superimposed ribbon diagrams of ATP-bound (magenta) and ATP-free (blue) hTRPV4-ARD. ATP is shown as sticks. (B) Superimposed Cα traces of human and chicken TRPV4-ARD. Finger 3 is twisted and shrunken in the ATP-bound (magenta) and ATP-unbound (green) hTRPV4-ARD structures, while the finger is extended in ATP-free hTRPV4-ARD (blue) and cTRPV4-ARD (gray). Several Finger 3 residues are disordered in three of six TRPV4-ARD structures. The structure of Finger 2 in the ATP-bound and -unbound forms differs from that in ATP-free forms.

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    Figure 2

    Figure 2. Aromatic residues on Fingers 2 and 3 have varied positions in hTRPV4-ARD structures. (A) The hTRPV4-ARD structures of ATP-bound (magenta), ATP-unbound (green), and ATP-free (blue) forms are superimposed. Aromatic residues are shown as sticks. (B) Detail of the Finger 2 and 3 loops. F272 and F273 on Finger 3 (black rectangles) are embedded in the aromatic cluster in the ATP-bound and -unbound forms but exposed in the ATP-free form. Y235 and Y236 on Finger 2 and Y281 and F282 on Finger 3 are located in similar positions but show variable orientations. F231, F282, Y283, and F284 show less variation.

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    Figure 3

    Figure 3. Structural comparison of TRPV-ARDs. (A) Superimposed main chain structure of TRPV-ARDs (rTRPV1-ARD, gray; rat and human TRPV2-ARD, cyan; ATP-bound hTRPV4-ARD, magenta; ATP-unbound hTRPV4-ARD, green; ATP-free hTRPV4-ARD, blue; and mouse TRPV6-ARD, black). Finger 3 and a part of Finger 2 are highly flexible. Several residues on Finger 3 are missing in one of two TRPV1-ARD structures and four of seven TRPV2-ARD structures. (B) ATP-binding site of hTRPV4-ARD and rTRPV1-ARD. Residues (sticks) within 4 Å of the ATP molecule and a surface map of the ATP-binding site in hTRPV4-ARD (left) and the corresponding residues in the rTRPV1-ARD ATP-binding site (right). The bBound ATP molecule is shown as sticks (orange and yellow). (C) Finger 2 (2) and Finger 3 (3) structures of ATP-bound rat TRPV1-ARD (gray), rat and human TRPV2-ARD (cyan), human ATP-bound TRPV4-ARD (magenta), and mouse TRPV6-ARD (black). (D) Aromatic residue positioned behind the adenine base of ATP in Finger 2 (F231 in human TRPV4-ARD). (E) This aromatic residue is conserved in TRPV-ARDs that bind ATP (red rectangle). (F and G) ATP-agarose pull-down assays for wild-type and mutant rTRPV1-ARD (F) or hTRPV4-ARD (G). Coomassie-stained gels (top) of wild-type and mutant proteins loaded (left) and bound to ATP-agarose in the absence (middle) or presence (right) of competing free ATP. The normalized intensity of protein recovered (mean ± SD; n = 3) is plotted below. The statistical significance of the change in binding to ATP-agarose with respect to the wild type (WT) was determined by a multiple-comparison test using Dunnett’s method, with p < 0.01 indicated by an asterisk.

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    Figure 4

    Figure 4. Effect of ATP on hTRPV4-ARD thermal stability. (A) Representative circular dichroism spectra of the purified TRPV4-ARD protein (3.4 μM) in the presence of ATP, AMP, or phosphate (1 mM each) at 10 °C. The wavelength (λ) of 222 nm used for thermostability assays is indicated by a vertical red line. (B) Representative traces of the thermostability assay. The molar ellipticity at 222 nm was measured as the protein solutions were heated at a rate of 1 °C/min. (C) Tm of TRPV4-ARD in the presence of 1 mM ATP, AMP, or phosphate. The statistical significance of the change in Tm was determined by a multiple-comparison test using the Tukey–Kramer method, with p < 0.05 and p < 0.01 indicated by one asterisk and two asterisks, respectively.

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    Figure 5

    Figure 5. Effect of ATP binding on protein stability in rTRPV1-ARD. (A) Structure of TRPV1-ARD (gray) bound to ATP (green, sticks), with buried Cys157 highlighted (spheres). (B) TRPV1-ARD is modified at cysteine residues by PEG-maleimide (mPEG), causing an electrophoretic mobility shift on a Coomassie-stained SDS gel. Abbreviations: WT, wild type; CL, a cysteine-less variant; C157, CL-TRPV1-ARD C157 single-cysteine variant. Shown is a representative Coomassie-stained gel from one of three experiments. (C) Time course for modification of single-cysteine TRPV1-ARD variants C157 and C362 with 0.5 mM mPEG at room temperature. (D) Data from four experiments like that depicted in panel C were quantified, and the mean ± standard deviation was plotted. (E and F) Molecular dynamics simulation in which the termini of the ATP-bound TRPV1-ARD (E) or TRPV1-ARD structure with ATP removed prior to equilibrating the system (F) are pulled apart at a rate of 20 nm/ns. Superimposed are the structures at the start (gold) and end (blue) of the simulations. (G and H) Root-mean-square deviation of each Cα atom over the course of the simulation mapped onto the starting models with (G) or without (H) ATP. The change in color from blue to red indicates changes in rmsd from 0 to 80 Å. Simulations in which the termini were pulled apart at a rate of 2 nm/ns gave similar results. See Table S3 of the Supporting Information for experimental details.

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    Figure 6

    Figure 6. Mutations associated with human diseases in hTRPV4-ARD. (A) Positions of mutations associated with human inherited diseases that lie within hTRPV4-ARD. Abbreviations: SEDM, spondyloepiphyseal dysplasia, type Maroteaux; SMDK, spondylometaphyseal dysplasia, type Kozolowski; MD, metatropic dysplasia; SMA, spinal muscular atrophy; SPMA, scapuloperoneal spinal muscular atrophy; CMTC2, Charcot-Marie-Tooth disease type 2C; HMSN2C, hereditary motor and sensory neuropathy 2C. This figure was inspired by ref 51. (B) Location of the disease-causing mutations within TRPV4-ARD. Shown as spheres are 12 residue positions at which a total of 15 mutations causing human inherited diseases have been identified. The ATP molecule is shown as sticks. Skeletal dysplasia and neurophathy mutations are depicted as green and blue spheres, respectively. (C) Leu199 is located at the hydrophobic interface between ANK2 and ANK3. (D) Glu183 and Arg232 form a salt bridge on the convex face of TRPV4-ARD.

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    Figure 7

    Figure 7. Thermal stability and ATP binding of hTRPV4-ARD mutants associated with inherited diseases. (A) The Tm determined by CD spectrometry in a phosphate-based buffer is plotted for wild-type and mutant hTRPV4-ARDs. The statistical significance is shown in Table S4 of the Supporting Information. (B) Coomassie-stained gels show wild-type and mutant TRPV4-ARDs loaded (top) and bound to ATP-agarose (bottom). (C) Normalized intensity of recovered protein (mean ± SD; n = 3). The statistical significance of the change in binding to ATP-agarose with respect to wild type (WT) was determined by a multiple-comparison test using Dunnett’s method, with p < 0.05 and p < 0.01 indicated by one asterisk and two asterisks, respectively.

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      A review. Capsaicin, the main ingredient in hot chili peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), is predicted to have 6 transmembrane (TM) domains and a short, pore-forming hydrophobic stretch between the 5th and 6th TM domains, and is activated not only by capsaicin but also by heat (>43°), acid, and various lipids. Within the TPRV1 protein, many regions and amino acids involved in specific functions (multimerization, capsaicin action, proton action, heat activation, desensitization, permeability, phosphorylation and modulation by lipids) have been identified since the cloning in 1997. Given the fact that TRPV1 is a key mol. in peripheral nociception, these regions and amino acids could prove useful for the development of novel anti-nociceptive or anti-inflammatory agents.
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      A review. The transient receptor potential vanilloid 4 (TRPV4) ion channel was named initially vanilloid-receptor-related osmotically activated channel (VR-OAC). Preliminary answers to the question, "What is the function of the trpv4 gene in live animals " are highlighted briefly in this review. In trpv4 null mice, TRPV4 is necessary for the maintenance of osmotic equil., and in Caenorhabditis elegans transgenic for mammalian TRPV4, TRPV4 directs the osmotic avoidance response in the context of the ASH "nociceptive" neuron. The mol. mechanisms of gating of TRPV4 in vivo need to be detd.; in particular, whether TRPV4 in live animals is gated via phosphorylation of defined amino-acid residues or more directly through the osmotic stimulus itself.
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      Thermosensation and pain
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      We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) superfamily. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and <17 degrees C for TRPA1), and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. TRPV2, TRPM8, and TRPA1 are also very likely to be involved in thermal nociception, because their activation thresholds are within the noxious range of temperatures.
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      The TRPV4 cation channel: A molecule linking skin temperature and barrier function
      Sokabe Takaaki; Tominaga Makoto
      Communicative & integrative biology (2010), 3 (6), 619-21 ISSN:.
      The skin barrier function is indispensable for terrestrial animals to avoid dehydration. The function is achieved by a hydrophobic cornified layer consisting of dead keratinocytes and lipids, and by an intercellular junction barrier formed among differentiated keratinocytes. A recent report demonstrated that TRPV4, one of the temperature-sensitive cation channels, contributes to the formation and maintenance of the intercellular junction-dependent barrier in the skin. TRPV4 associates with the E-cadherin complex via β-catenin, and thereby participates in the promotion of cell-cell junction development. TRPV4 allows influx of Ca(2+) ions from the extracellular space at physiological skin temperatures. The Ca(2+) influx induces Rho activation and promotes actin fiber organization and junction formation, thereby augmenting barrier integrity. Indeed, the intercellular junction structures and the skin barrier function were impaired in TRPV4-deficeint mice. This novel role of TRPV4 in keratinocytes may explain the significant correlation between temperature and the condition of skin.>
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      Effects of body temperature on neural activity in the hippocampus: regulation of resting membrane potentials by transient receptor potential vanilloid 4
      Shibasaki, Koji; Suzuki, Makoto; Mizuno, Atsuko; Tominaga, Makoto
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      Physiol. body temp. is an important determinant for neural functions, and it is well established that changes in temp. have dynamic influences on hippocampal neural activities. However, the detailed mol. mechanisms have never been clarified. Here, we show that hippocampal neurons express functional transient receptor potential vanilloid 4 (TRPV4), one of the thermosensitive TRP (transient receptor potential) channels, and that TRPV4 is constitutively active at physiol. temp. Activation of TRPV4 at 37°C depolarized the resting membrane potential in hippocampal neurons by allowing cation influx, which was obsd. in wild-type (WT) neurons, but not in TRPV4-deficient (TRPV4KO) cells, although dendritic morphol., synaptic marker clustering, and synaptic currents were indistinguishable between the two genotypes. Furthermore, current injection studies revealed that TRPV4KO neurons required larger depolarization to evoke firing, equiv. to WT neurons, indicating that TRPV4 is a key regulator for hippocampal neural excitabilities. We conclude that TRPV4 is activated by physiol. temp. in hippocampal neurons and thereby controls their excitability.
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      Alessandri-Haber, N., Dina, O. A., Joseph, E. K., Reichling, D., and Levine, J. D. (2006) A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators J. Neurosci. 26, 3864– 3874
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      21
      A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators
      Alessandri-Haber, Nicole; Dina, Olayinka A.; Joseph, Elizabeth K.; Reichling, David; Levine, Jon D.
      Journal of Neuroscience (2006), 26 (14), 3864-3874CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      The transient receptor potential vanilloid 4 (TRPV4) is a primary afferent transducer that plays a crucial role in neuropathic hyperalgesia for osmotic and mech. stimuli, as well as in inflammatory mediator-induced hyperalgesia for osmotic stimuli. In view of the clin. importance of mech. hyperalgesia in inflammatory states, the present study investigated the role of TRPV4 in mech. hyperalgesia induced by inflammatory mediators and the second-messenger pathways involved. Intradermal injection of either the inflammogen carrageenan or a soup of inflammatory mediators enhanced the nocifensive paw-withdrawal reflex elicited by hypotonic or mech. stimuli in rat. Spinal administration of TRPV4 antisense oligodeoxynucleotide blocked the enhancement without altering baseline nociceptive threshold. Similarly, in TRPV4-/- knock-out mice, inflammatory soup failed to induce any significant mech. or osmotic hyperalgesia. In vitro investigation showed that inflammatory mediators engage the TRPV4-mediated mechanism of sensitization by direct action on dissocd. primary afferent neurons. Addnl. behavioral observations suggested that multiple mediators are necessary to achieve sufficient activation of the cAMP pathway to engage the TRPV4-dependent mechanism of hyperalgesia. In addn., direct activation of protein kinase A or protein kinase C ε, two pathways that mediate inflammation-induced mech. hyperalgesia, also induced hyperalgesia for both hypotonic and mech. stimuli that was decreased by TRPV4 antisense and absent in TRPV4-/- mice. We conclude that TRPV4 plays a crucial role in the mech. hyperalgesia that is generated by the concerted action of inflammatory mediators present in inflamed tissues.
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      Masuyama, R., Vriens, J., Voets, T., Karashima, Y., Owsianik, G., Vennekens, R., Lieben, L., Torrekens, S., Moermans, K., Vanden Bosch, A., Bouillon, R., Nilius, B., and Carmeliet, G. (2008) TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts Cell Metab. 8, 257– 265
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      22
      TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts
      Masuyama, Ritsuko; Vriens, Joris; Voets, Thomas; Karashima, Yuji; Owsianik, Grzegorz; Vennekens, Rudi; Lieben, Liesbet; Torrekens, Sophie; Moermans, Karen; Vanden Bosch, An; Bouillon, Roger; Nilius, Bernd; Carmeliet, Geert
      Cell Metabolism (2008), 8 (3), 257-265CODEN: CMEEB5; ISSN:1550-4131. (Cell Press)
      Calcium signaling controls multiple cellular functions and is regulated by the release from internal stores and entry from extracellular fluid. In bone, osteoclast differentiation is induced by RANKL (receptor activator of NF-κB ligand)-evoked intracellular Ca2+ oscillations, which trigger nuclear factor-activated T cells (NFAT) c1-responsive gene transcription. However, the Ca2+ channels involved remain largely unidentified. Here we show that genetic ablation in mice of Trpv4, a Ca2+-permeable channel of the transient receptor potential (TRP) family, increases bone mass by impairing bone resorption. TRPV4 mediates basolateral Ca2+ influx specifically in large osteoclasts when Ca2+ oscillations decline. TRPV4-mediated Ca2+ influx hereby secures intracellular Ca2+ concns., ensures NFATc1-regulated gene transcription, and regulates the terminal differentiation and activity of osteoclasts. In conclusion, our data indicate that Ca2+ oscillations and TRPV4-mediated Ca2+ influx are sequentially required to sustain NFATc1-dependent gene expression throughout osteoclast differentiation, and we propose TRPV4 as a therapeutic target for bone diseases.
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      Transient receptor potential channelopathies
      Nilius, Bernd; Owsianik, Grzegorz
      Pfluegers Archiv (2010), 460 (2), 437-450CODEN: PFLABK; ISSN:0031-6768. (Springer GmbH)
      A review. In the past years, several hereditary diseases caused by defects in transient receptor potential channels (TRP) genes have been described. This review summarizes our current knowledge about TRP channelopathies and their possible pathomechanisms. Based on available genetic indications, we will also describe several putative pathol. conditions in which (mal)function of TRP channels could be anticipated.
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      Verma, P., Kumar, A., and Goswami, C. (2010) TRPV4-mediated channelopathies Channels 4, 319– 328
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      Rock, M. J., Prenen, J., Funari, V. A., Funari, T. L., Merriman, B., Nelson, S. F., Lachman, R. S., Wilcox, W. R., Reyno, S., Quadrelli, R., Vaglio, A., Owsianik, G., Janssens, A., Voets, T., Ikegawa, S., Nagai, T., Rimoin, D. L., Nilius, B., and Cohn, D. H. (2008) Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia Nat. Genet. 40, 999– 1003
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      25
      Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia
      Rock, Matthew J.; Prenen, Jean; Funari, Vincent A.; Funari, Tara L.; Merriman, Barry; Nelson, Stanley F.; Lachman, Ralph S.; Wilcox, William R.; Reyno, Soraya; Quadrelli, Roberto; Vaglio, Alicia; Owsianik, Grzegorz; Janssens, Annelies; Voets, Thomas; Ikegawa, Shiro; Nagai, Toshiro; Rimoin, David L.; Nilius, Bernd; Cohn, Daniel H.
      Nature Genetics (2008), 40 (8), 999-1003CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
      Daniel Cohn and colleagues identify mutations in the gene encoding the calcium-permeable cation channel TRPV4 in families with autosomal dominant brachyolmia. Functional studies show that the mutations result in gain-of-function of channel activation. Daniel Cohn and colleagues identify mutations in the gene encoding the calcium-permeable cation channel TRPV4 in families with autosomal dominant brachyolmia. Functional studies show that the mutations result in gain-of-function of channel activation. The brachyolmias constitute a clin. and genetically heterogeneous group of skeletal dysplasias characterized by a short trunk, scoliosis and mild short stature. Here, we identify a locus for an autosomal dominant form of brachyolmia on chromosome 12q24.1-12q24.2. Among the genes in the genetic interval, we selected TRPV4, which encodes a calcium permeable cation channel of the transient receptor potential (TRP) vanilloid family, as a candidate gene because of its cartilage-selective gene expression pattern. In two families with the phenotype, we identified point mutations in TRPV4 that encoded R616Q and V620I substitutions, resp. Patch clamp studies of transfected HEK cells showed that both mutations resulted in a dramatic gain of function characterized by increased constitutive activity and elevated channel activation by either mechano-stimulation or agonist stimulation by arachidonic acid or the TRPV4-specific agonist 4α-phorbol 12,13-didecanoate (4αPDD). This study thus defines a previously unknown mechanism, activation of a calcium-permeable TRP ion channel, in skeletal dysplasia pathogenesis.
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    26. 26
      Krakow, D., Vriens, J., Camacho, N., Luong, P., Deixler, H., Funari, T. L., Bacino, C. A., Irons, M. B., Holm, I. A., Sadler, L., Okenfuss, E. B., Janssens, A., Voets, T., Rimoin, D. L., Lachman, R. S., Nilius, B., and Cohn, D. H. (2009) Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia Am. J. Hum. Genet. 84, 307– 315
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      26
      Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia
      Krakow, Deborah; Vriens, Joris; Camacho, Natalia; Luong, Phi; Deixler, Hannah; Funari, Tara L.; Bacino, Carlos A.; Irons, Mira B.; Holm, Ingrid A.; Sadler, Laurie; Okenfuss, Ericka B.; Janssens, Annelies; Voets, Thomas; Rimoin, David L.; Lachman, Ralph S.; Nilius, Bernd; Cohn, Daniel H.
      American Journal of Human Genetics (2009), 84 (3), 307-315CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      The spondylometaphyseal dysplasias (SMDs) are a group of short-stature disorders distinguished by abnormalities in the vertebrae and the metaphyses of the tubular bones. SMD Kozlowski type (SMDK) is a well-defined autosomal-dominant SMD characterized by significant scoliosis and mild metaphyseal abnormalities in the pelvis. The vertebrae exhibit platyspondyly and overfaced pedicles similar to autosomal-dominant brachyolmia, which can result from heterozygosity for activating mutations in the gene encoding TRPV4, a calcium-permeable ion channel. Mutation anal. in six out of six patients with SMDK demonstrated heterozygosity for missense mutations in TRPV4, and one mutation, predicting a R594H substitution, was recurrent in four patients. Similar to autosomal-dominant brachyolmia, the mutations altered basal calcium channel activity in vitro. Metatropic dysplasia is another SMD that has been proposed to have both clin. and genetic heterogeneity. Patients with the nonlethal form of metatropic dysplasia present with a progressive scoliosis, widespread metaphyseal involvement of the appendicular skeleton, and carpal ossification delay. Because of some similar radiog. features between SMDK and metatropic dysplasia, TRPV4 was tested as a disease gene for nonlethal metatropic dysplasia. In two sporadic cases, heterozygosity for de novo missense mutations in TRPV4 was found. The findings demonstrate that mutations in TRPV4 produce a phenotypic spectrum of skeletal dysplasias from the mild autosomal-dominant brachyolmia to SMDK to autosomal-dominant metatropic dysplasia, suggesting that these disorders should be grouped into a new bone dysplasia family.
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      Deng, H. X., Klein, C. J., Yan, J., Shi, Y., Wu, Y., Fecto, F., Yau, H. J., Yang, Y., Zhai, H., Siddique, N., Hedley-Whyte, E. T., Delong, R., Martina, M., Dyck, P. J., and Siddique, T. (2010) Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4 Nat. Genet. 42, 165– 169
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      27
      Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4
      Deng, Han-Xiang; Klein, Christopher J.; Yan, Jian-Hua; Shi, Yong; Wu, Yan-Hong; Fecto, Faisal; Yau, Hau-Jie; Yang, Yi; Zhai, Hong; Siddique, Nailah; Hedley-Whyte, E. Tessa; De Long, Robert; Martina, Marco; Dyck, Peter J.; Siddique, Teepu
      Nature Genetics (2010), 42 (2), 165-169CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
      Scapuloperoneal spinal muscular atrophy (SPSMA) and hereditary motor and sensory neuropathy type IIC (HMSN IIC, also known as HMSN2C or Charcot-Marie-Tooth disease type 2C (CMT2C)) are phenotypically heterogeneous disorders involving topog. distinct nerves and muscles. We originally described a large New England family of French-Canadian origin with SPSMA and an American family of English and Scottish descent with CMT2C. We mapped SPSMA and CMT2C risk loci to 12q24.1-q24.31 with an overlapping region between the two diseases. Further anal. reduced the CMT2C risk locus to a 4-Mb region. Here we report that SPSMA and CMT2C are allelic disorders caused by mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 4 (TRPV4). Functional anal. revealed that increased calcium channel activity is a distinct property of both SPSMA- and CMT2C-causing mutant proteins. Our findings link mutations in TRPV4 to altered calcium homeostasis and peripheral neuropathies, implying a pathogenic mechanism and possible options for therapy for these disorders.
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    28. 28
      Landoure, G., Zdebik, A. A., Martinez, T. L., Burnett, B. G., Stanescu, H. C., Inada, H., Shi, Y., Taye, A. A., Kong, L., Munns, C. H., Choo, S. S., Phelps, C. B., Paudel, R., Houlden, H., Ludlow, C. L., Caterina, M. J., Gaudet, R., Kleta, R., Fischbeck, K. H., and Sumner, C. J. (2010) Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C Nat. Genet. 42, 170– 174
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      28
      Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C
      Landoure, Guida; Zdebik, Anselm A.; Martinez, Tara L.; Burnett, Barrington G.; Stanescu, Horia C.; Inada, Hitoshi; Shi, Yi-Jun; Taye, Addis A.; Kong, Ling-Ling; Munns, Clare H.; Choo, Shelly S.; Phelps, Christopher B.; Paudel, Reema; Houlden, Henry; Ludlow, Christy L.; Caterina, Michael J.; Gaudet, Rachelle; Kleta, Robert; Fischbeck, Kenneth H.; Sumner, Charlotte J.
      Nature Genetics (2010), 42 (2), 170-174CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)
      Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously assocd. with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-assocd. mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.
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    29. 29
      Jin, X., Touhey, J., and Gaudet, R. (2006) Structure of the N-terminal ankyrin repeat domain of the TRPV2 ion channel J. Biol. Chem. 281, 25006– 25010
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    30. 30
      Otwinowski, Z. and Minor, W. (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode Methods Enzymol. 276, 307– 326
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      30
      Processing of x-ray diffraction data collected in oscillation mode
      Otwinowski, Zbyszek; Minor, Wladek
      Methods in Enzymology (1997), 276 (Macromolecular Crystallography, Part A), 307-326CODEN: MENZAU; ISSN:0076-6879. (Academic)
      Macromol. crystallog. is an iterative process. Rarely do the first crystals provide all the necessary data to solve the biol. problem being studied. Each step benefits from experience learned in previous steps. To monitor the progress, the HKL package provides 2 tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), are provided, and the Bayesian reasoning and multicomponent error model facilitates obtaining the proper error ests. and (2) visualization of the process plays a double role by helping the operator to confirm that the process of data redn., including the resulting statistics, is correct, and allowing one to evaluate problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and redn. has been reached. At that point, the effort should be directed to solving the structure. The methods presented here have been applied to solve a large variety of problems, from inorg. mols. with 5 Å unit cell to rotavirus of 700 Å diam. crystd. in 700 × 1000 × 1400 Å cell. Overall quality of the method was tested by many researchers by successful application of the programs to MAD structure detns.
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    31. 31
      Vagin, A. and Teplyakov, A. (2000) An approach to multi-copy search in molecular replacement Acta Crystallogr. D56, 1622– 1624
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    32. 32
      McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software J. Appl. Crystallogr. 40, 658– 674
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      32
      Phaser crystallographic software
      McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.
      Journal of Applied Crystallography (2007), 40 (4), 658-674CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
      Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
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    33. 33
      Emsley, P. and Cowtan, K. (2004) Coot: Model-building tools for molecular graphics Acta Crystallogr. D60, 2126– 2132
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      33
      Coot: model-building tools for molecular graphics
      Emsley, Paul; Cowtan, Kevin
      Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
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    34. 34
      Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr. D53, 240– 255
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      34
      Refinement of macromolecular structures by the maximum-likelihood method
      Murshudov, Garib N.; Vagin, Alexei A.; Dodson, Eleanor J.
      Acta Crystallographica, Section D: Biological Crystallography (1997), D53 (3), 240-255CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard)
      A review with many refs. on the math. basis of max. likelihood. The likelihood function for macromol. structures is extended to include prior phase information and exptl. std. uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estg. σA using "free" reflections is described and its effects analyzed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the max.-likelihood residual are consistently better than those obtained from least-squares refinement.
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    35. 35
      Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: Visual molecular dynamics J. Mol. Graphics 14, 27– 38
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    36. 36
      Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable molecular dynamics with NAM J. Comput. Chem. 26, 1781– 1802
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    37. 37
      MacKerell, A. D., Jr., Bashford, D., Bellott, M., Dunbrack, R. L., Jr., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., III, Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiórkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins J. Phys. Chem. B 102, 3586– 3616
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      37
      All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins
      MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E., III; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M.
      Journal of Physical Chemistry B (1998), 102 (18), 3586-3616CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
      New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used exptl. gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the at. charges, were detd. by fitting ab initio interaction energies and geometries of complexes between water and model compds. that represented the backbone and the various side chains. In addn., dipole moments, exptl. heats and free energies of vaporization, solvation and sublimation, mol. vols., and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in a crystal. A detailed anal. of the relationship between the alanine dipeptide potential energy surface and calcd. protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in soln. and in crystals. Extensive comparisons between mol. dynamics simulation and exptl. data for polypeptides and proteins were performed for both structural and dynamic properties. Calcd. data from energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with exptl. crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of mols. of biol. interest.
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    38. 38
      Mackerell, A. D., Jr., Feig, M., and Brooks, C. L., III (2004) Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations J. Comput. Chem. 25, 1400– 1415
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      38
      Extending the treatment of backbone energetics in protein force fields: Limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations
      MacKerell, Alexander D., Jr.; Feig, Michael; Brooks, Charles L., III
      Journal of Computational Chemistry (2004), 25 (11), 1400-1415CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      Computational studies of proteins based on empirical force fields represent a powerful tool to obtain structure-function relationships at an at. level, and are central in current efforts to solve the protein folding problem. The results from studies applying these tools are, however, dependent on the quality of the force fields used. In particular, accurate treatment of the peptide backbone is crucial to achieve representative conformational distributions in simulation studies. To improve the treatment of the peptide backbone, quantum mech. (QM) and mol. mech. (MM) calcns. were undertaken on the alanine, glycine, and proline dipeptides, and the results from these calcns. were combined with mol. dynamics (MD) simulations of proteins in crystal and aq. environments. QM potential energy maps of the alanine and glycine dipeptides at the LMP2/cc-pVxZ/MP2/6-31G* levels, where x = D, T, and Q, were detd., and are compared to available QM studies on these mols. The LMP2/cc pVQZ//MP2/6-31G* energy surfaces for all three dipeptides were then used to improve the MM treatment of the dipeptides. These improvements included addnl. parameter optimization via Monte Carlo simulated annealing and extension of the potential energy function to contain peptide backbone .vphi., ψ dihedral crossterms or a .vphi., ψ grid-based energy correction term. Simultaneously, MD simulations of up to seven proteins in their cryst. environments were used to validate the force field enhancements. Comparison with QM and crystallog. data showed that an addnl. optimization of the .vphi., ψ dihedral parameters along with the grid-based energy correction were required to yield significant improvements over the CHARMM22 force field. However, systematic deviations in the treatment of .vphi. and ψ in the helical and sheet regions were evident. Accordingly, empirical adjustments were made to the grid-based energy correction for alanine and glycine to account for these systematic differences. These adjustments lead to greater deviations from QM data for the two dipeptides but also yielded improved agreement with exptl. crystallog. data. These improvements enhance the quality of the CHARMM force field in treating proteins. This extension of the potential energy function is anticipated to facilitate improved treatment of biol. macromols. via MM approaches in general.
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    39. 39
      McCleverty, C. J., Koesema, E., Patapoutian, A., Lesley, S. A., and Kreusch, A. (2006) Crystal structure of the human TRPV2 channel ankyrin repeat domain Protein Sci. 15, 2201– 2206
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      39
      Crystal structure of the human TRPV2 channel ankyrin repeat domain
      McCleverty, Clare J.; Koesema, Eric; Patapoutian, Ardem; Lesley, Scott A.; Kreusch, Andreas
      Protein Science (2006), 15 (9), 2201-2206CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
      TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain 3-4 ankyrin repeats. Here, the authors report the 1st structure from the TRPV channel subfamily, a 1.7-Å resoln. crystal structure of human TRPV2 ARD. The crystal structure revealed a 6-ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contained extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last 2 repeats. The TRPV2 ARD provided the 1st structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.
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    40. 40
      Phelps, C. B., Huang, R. J., Lishko, P. V., Wang, R. R., and Gaudet, R. (2008) Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels Biochemistry 47, 2476– 2484
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      Croy, C. H., Bergqvist, S., Huxford, T., Ghosh, G., and Komives, E. A. (2004) Biophysical characterization of the free IκBα ankyrin repeat domain in solution Protein Sci. 13, 1767– 1777
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      41
      Biophysical characterization of the free IκBα ankyrin repeat domain in solution
      Croy, Carrie Hughes; Bergqvist, Simon; Huxford, Tom; Ghosh, Gourisankar; Komives, Elizabeth A.
      Protein Science (2004), 13 (7), 1767-1777CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
      The crystal structure of IκBα in complex with the transcription factor, nuclear factor κ-B (NF-κB) shows six ankyrin repeats, which are all ordered. Electron d. was not obsd. for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IκBα (67-317) when it is not in complex with NF-κB, we have carried out CD spectroscopy, 8-anilino-1-naphthalenesulfonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange expts. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amt. of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange expts. show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. IκBα unfolds with a Tm of 42°, and forms a sol. aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biol. function of IκBα are discussed.
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    42. 42
      Truhlar, S. M., Torpey, J. W., and Komives, E. A. (2006) Regions of IκBα that are critical for its inhibition of NF-κB·DNA interaction fold upon binding to NF-κB Proc. Natl. Acad. Sci. U.S.A. 103, 18951– 18956
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      Barrick, D., Ferreiro, D. U., and Komives, E. A. (2008) Folding landscapes of ankyrin repeat proteins: Experiments meet theory Curr. Opin. Struct. Biol. 18, 27– 34
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      43
      Folding landscapes of ankyrin repeat proteins: Experiments meet theory
      Barrick, Doug; Ferreiro, Diego U.; Komives, Elizabeth A.
      Current Opinion in Structural Biology (2008), 18 (1), 27-34CODEN: COSBEF; ISSN:0959-440X. (Elsevier B.V.)
      A review. Nearly 6% of eukaryotic protein sequences contain ankyrin repeat (AR) domains, which consist of several repeats and often function in binding. AR proteins show highly cooperative folding despite a lack of long-range contacts. Both theory and expt. converge to explain that formation of the interface between elements is more favorable than formation of any individual repeat unit. IκBα and Notch both undergo partial folding upon binding perhaps influencing the binding free energy. The simple architecture, combined with identification of consensus residues that are important for stability, has enabled systematic perturbation of the energy landscape by single point mutations that affect stability or by addn. of consensus repeats. The folding energy landscapes appear highly plastic, with small perturbations re-routing folding pathways.
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    44. 44
      Salazar, H., Llorente, I., Jara-Oseguera, A., Garcia-Villegas, R., Munari, M., Gordon, S. E., Islas, L. D., and Rosenbaum, T. (2008) A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic Nat. Neurosci. 11, 255– 261
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      44
      A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic
      Salazar, Hector; Llorente, Itzel; Jara-Oseguera, Andres; Garcia-Villegas, Refugio; Munari, Mika; Gordon, Sharona E.; Islas, Leon D.; Rosenbaum, Tamara
      Nature Neuroscience (2008), 11 (3), 255-261CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
      Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compds. found in garlic, onion, mustard and cinnamon exts., activation of TRPV1 by these exts. remains controversial. Here the authors establish that TRPV1 is activated by pungent exts. from onion and garlic, as well as by allicin, the active compd. in these prepns., and participates together with TRPA1 in the pain-related behavior induced by this compd. The authors found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects the authors obsd. The authors findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the mol. basis of noxious stimuli detection.
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    45. 45
      Isralewitz, B., Gao, M., and Schulten, K. (2001) Steered molecular dynamics and mechanical functions of proteins Curr. Opin. Struct. Biol. 11, 224– 230
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      45
      Steered molecular dynamics and mechanical functions of proteins
      Isralewitz, Barry; Gao, Mu; Schulten, Klaus
      Current Opinion in Structural Biology (2001), 11 (2), 224-230CODEN: COSBEF; ISSN:0959-440X. (Elsevier Science Ltd.)
      A review with 50 refs. At. force microscopy of single mols., steered mol. dynamics and the theory of stochastic processes have established a new field that investigates mech. functions of proteins, such as ligand-receptor binding/unbinding and elasticity of muscle proteins during stretching. The combination of these methods yields information on the energy landscape that controls mech. function and on the force-bearing components of proteins, as well as on the underlying phys. mechanisms.
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    46. 46
      Camacho, N., Krakow, D., Johnykutty, S., Katzman, P. J., Pepkowitz, S., Vriens, J., Nilius, B., Boyce, B. F., and Cohn, D. H. (2010) Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia Am. J. Med. Genet., Part A 152A, 1169– 1177
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      46
      Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia
      Camacho, Natalia; Krakow, Deborah; Johnykutty, Sharlin; Katzman, Philip J.; Pepkowitz, Samuel; Vriens, Joris; Nilius, Bernd; Boyce, Brendan F.; Cohn, Daniel H.
      American Journal of Medical Genetics, Part A (2010), 152A (5), 1169-1177CODEN: AJMGB8; ISSN:1552-4825. (Wiley-Liss, Inc.)
      Metatropic dysplasia is a clin. heterogeneous skeletal dysplasia characterized by short extremities, a short trunk with progressive kyphoscoliosis, and craniofacial abnormalities that include a prominent forehead, midface hypoplasia, and a squared-off jaw. Dominant mutations in the gene encoding TRPV4, a calcium permeable ion channel, were identified all 10 of a series of metatropic dysplasia cases, ranging in severity from mild to perinatal lethal. These data demonstrate that the lethal form of the disorder is dominantly inherited and suggest locus homogeneity in the disease. Electrophysiol. studies demonstrated that the mutations activate the channel, indicating that the mechanism of disease may result from increased calcium in chondrocytes. Histol. studies in two cases of lethal metatropic dysplasia revealed markedly disrupted endochondral ossification, with reduced nos. of hypertrophic chondrocytes and presence of islands of cartilage within the zone of primary mineralization. These data suggest that altered chondrocyte differentiation in the growth plate leads to the clin. findings in metatropic dysplasia.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnsFylurg%253D&md5=00b52e12ff7d4602a43cf7270dc2384b
    47. 47
      Dai, J., Kim, O. H., Cho, T. J., Schmidt-Rimpler, M., Tonoki, H., Takikawa, K., Haga, N., Miyoshi, K., Kitoh, H., Yoo, W. J., Choi, I. H., Song, H. R., Jin, D. K., Kim, H. T., Kamasaki, H., Bianchi, P., Grigelioniene, G., Nampoothiri, S., Minagawa, M., Miyagawa, S. I., Fukao, T., Marcelis, C., Jansweijer, M. C., Hennekam, R. C., Bedeschi, F., Mustonen, A., Jiang, Q., Ohashi, H., Furuichi, T., Unger, S., Zabel, B., Lausch, E., Superti-Furga, A., Nishimura, G., and Ikegawa, S. (2010) Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family J. Med. Genet. 47, 704– 709
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      47
      Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family
      Dai, J.; Kim, O.-H.; Cho, T.-J.; Schmidt-Rimpler, M.; Tonoki, H.; Takikawa, K.; Haga, N.; Miyoshi, K.; Kitoh, H.; Yoo, W.-J.; Choi, I.-H.; Song, H.-R.; Jin, D.-K.; Kim, H.-T.; Kamasaki, H.; Bianchi, P.; Grigelioniene, G.; Nampoothiri, S.; Minagawa, M.; Miyagawa, S.-i.; Fukao, T.; Marcelis, C.; Jansweijer, M. C. E.; Hennekam, R. C. M.; Bedeschi, F.; Mustonen, A.; Jiang, Q.; Ohashi, H.; Furuichi, T.; Unger, S.; Zabel, B.; Lausch, E.; Superti-Furga, A.; Nishimura, G.; Ikegawa, S.
      Journal of Medical Genetics (2010), 47 (10), 704-709CODEN: JMDGAE; ISSN:0022-2593. (BMJ Publishing Group)
      Mutations in TRPV4, a gene that encodes a Ca2+ permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. To examine TRPV4 mutation spectrum and phenotype-genotype assocn., we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471 del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An assocn. between the position of mutations and the disease phenotype was also obsd. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been obsd. at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic labs. establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.
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    48. 48
      Nishimura, G., Dai, J., Lausch, E., Unger, S., Megarbane, A., Kitoh, H., Kim, O. H., Cho, T. J., Bedeschi, F., Benedicenti, F., Mendoza-Londono, R., Silengo, M., Schmidt-Rimpler, M., Spranger, J., Zabel, B., Ikegawa, S., and Superti-Furga, A. (2010) Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2), and parastremmatic dysplasia are caused by TRPV4 mutations Am. J. Med. Genet., Part A 152A, 1443– 1449
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      48
      Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2), and parastremmatic dysplasia are caused by TRPV4 mutations
      Nishimura Gen; Dai Jin; Lausch Ekkehart; Unger Sheila; Megarbane Andre; Kitoh Hiroshi; Kim Ok Hwa; Cho Tae-Joon; Bedeschi Francesca; Benedicenti Francesco; Mendoza-Londono Roberto; Silengo Margherita; Schmidt-Rimpler Maren; Spranger Jurgen; Zabel Bernhard; Ikegawa Shiro; Superti-Furga Andrea
      American journal of medical genetics. Part A (2010), 152A (6), 1443-9 ISSN:.
      Recent discoveries have established the existence of a family of skeletal dysplasias caused by dominant mutations in TRPV4. This family comprises, in order of increasing severity, dominant brachyolmia, spondylo-metaphyseal dysplasia Kozlowski type, and metatropic dysplasia. We tested the hypothesis that a further condition, Spondylo-epiphyseal dysplasia (SED), Maroteaux type (MIM 184095; also known as pseudo-Morquio syndrome type 2), could be caused by TRPV4 mutations. We analyzed six individuals with Maroteaux type SED, including three who had previously been reported. All six patients were found to have heterozygous TRPV4 mutations; three patients had unreported mutations, while three patients had mutations previously described in association with metatropic dysplasia. In addition, we tested one individual with a distinct rare disorder, parastremmatic dysplasia (MIM 168400). This patient had a common, recurrent mutation seen in several patients with Kozlowski type spondylo-metaphyseal dysplasia. We conclude that SED Maroteaux type and parastremmatic dysplasia are part of the TRPV4 dysplasia family and that TRPV4 mutations show considerable variability in phenotypic expression resulting in distinct clinical-radiographic phenotypes.
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    49. 49
      Zimon, M., Baets, J., Auer-Grumbach, M., Berciano, J., Garcia, A., Lopez-Laso, E., Merlini, L., Hilton-Jones, D., McEntagart, M., Crosby, A. H., Barisic, N., Boltshauser, E., Shaw, C. E., Landoure, G., Ludlow, C. L., Gaudet, R., Houlden, H., Reilly, M. M., Fischbeck, K. H., Sumner, C. J., Timmerman, V., Jordanova, A., and Jonghe, P. D. (2010) Dominant mutations in the cation channel gene transient receptor potential vanilloid 4 cause an unusual spectrum of neuropathies Brain 133, 1798– 1809
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      There is no corresponding record for this reference.
    50. 50
      Clapham, D. E. and Miller, C. (2011) A thermodynamic framework for understanding temperature sensing by transient receptor potential (TRP) channels Proc. Natl. Acad. Sci. U.S.A. 108, 19492– 19497
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      There is no corresponding record for this reference.
    51. 51
      Dai, J., Cho, T. J., Unger, S., Lausch, E., Nishimura, G., Kim, O. H., Superti-Furga, A., and Ikegawa, S. (2010) TRPV4-pathy, a novel channelopathy affecting diverse systems J. Hum. Genet. 55, 400– 402
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      51
      TRPV4-pathy, a novel channelopathy affecting diverse systems
      Dai Jin; Cho Tae-Joon; Unger Sheila; Lausch Ekkehart; Nishimura Gen; Kim Ok-Hwa; Superti-Furga Andrea; Ikegawa Shiro
      Journal of human genetics (2010), 55 (7), 400-2 ISSN:.
      Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a calcium-permeable nonselective cation channel of unknown biological function. TRPV4 mutation was first identified in brachyolmia, and then in a spectrum of autosomal-dominant skeletal dysplasias, which includes Kozlowski type of spondylometaphyseal dysplasia, metatropic dysplasia, Maroteaux type of spondyloepiphyseal dysplasia and parastremmatic dysplasia. Recently, TRPV4 mutation has also been identified in a spectrum of neuromuscular diseases that includes congenital distal spinal muscular atrophy (SMA), scapuloperoneal SMA, and hereditary motor and sensory neuropathy type IIC. These diverse spectrums of diseases compose a novel channelopathy, TRPV4-pathy, which could further include polygenic traits such as serum sodium concentration and a chronic obstructive pulmonary disease. In this review, we clarified the TRPV4 mutation spectrum, and discussed the phenotypic complexity of TRPV4-pathy and its pathogenic mechanisms. TRPV4-pathy may extend further to other monogenic and polygenic diseases.
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    52. 52
      Andreucci, E., Aftimos, S., Alcausin, M., Haan, E., Hunter, W., Kannu, P., Kerr, B., McGillivray, G., Gardner, R. M., Patricelli, M. G., Sillence, D., Thompson, E., Zacharin, M., Zankl, A., Lamande, S. R., and Savarirayan, R. (2011) TRPV4 related skeletal dysplasias: A phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families Orphanet. J. Rare Dis. 6, 37
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      52
      TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families
      Andreucci Elena; Aftimos Salim; Alcausin Melanie; Haan Eric; Hunter Warwick; Kannu Peter; Kerr Bronwyn; McGillivray George; McKinlay Gardner R J; Patricelli Maria G; Sillence David; Thompson Elizabeth; Zacharin Margaret; Zankl Andreas; Lamande Shireen R; Savarirayan Ravi
      Orphanet journal of rare diseases (2011), 6 (), 37 ISSN:.
      BACKGROUND: The TRPV4 gene encodes a calcium-permeable ion-channel that is widely expressed, responds to many different stimuli and participates in an extraordinarily wide range of physiologic processes. Autosomal dominant brachyolmia, spondylometaphyseal dysplasia Kozlowski type (SMDK) and metatropic dysplasia (MD) are currently considered three distinct skeletal dysplasias with some shared clinical features, including short stature, platyspondyly, and progressive scoliosis. Recently, TRPV4 mutations have been found in patients diagnosed with these skeletal phenotypes. METHODS AND RESULTS: We critically analysed the clinical and radiographic data on 26 subjects from 21 families, all of whom had a clinical diagnosis of one of the conditions described above: 15 with MD; 9 with SMDK; and 2 with brachyolmia. We sequenced TRPV4 and identified 9 different mutations in 22 patients, 4 previously described, and 5 novel. There were 4 mutation-negative cases: one with MD and one with SMDK, both displaying atypical clinical and radiographic features for these diagnoses; and two with brachyolmia, who had isolated spine changes and no metaphyseal involvement. CONCLUSIONS: Our data suggest the TRPV4 skeletal dysplasias represent a continuum of severity with areas of phenotypic overlap, even within the same family. We propose that AD brachyolmia lies at the mildest end of this spectrum and, since all cases described with this diagnosis and TRPV4 mutations display metaphyseal changes, we suggest that it is not a distinct entity but represents the mildest phenotypic expression of SMDK.
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    53. 53
      Klein, C. J., Shi, Y., Fecto, F., Donaghy, M., Nicholson, G., McEntagart, M. E., Crosby, A. H., Wu, Y., Lou, H., McEvoy, K. M., Siddique, T., Deng, H. X., and Dyck, P. J. (2011) TRPV4 mutations and cytotoxic hypercalcemia in axonal Charcot-Marie-Tooth neuropathies Neurology 76, 887– 894
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      There is no corresponding record for this reference.
    54. 54
      Fecto, F., Shi, Y., Huda, R., Martina, M., Siddique, T., and Deng, H. X. (2011) Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies J. Biol. Chem. 286, 17281– 17291
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      There is no corresponding record for this reference.
    55. 55
      Loukin, S., Su, Z., and Kung, C. (2011) Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations PLoS One 6, e19533
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      There is no corresponding record for this reference.
    56. 56
      Schaefer, M. (2005) Homo- and heteromeric assembly of TRP channel subunits Pfluegers Arch. 451, 35– 42
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      56
      Homo- and heteromeric assembly of TRP channel subunits
      Schaefer, Michael
      Pfluegers Archiv (2005), 451 (1), 35-42CODEN: PFLABK; ISSN:0031-6768. (Springer GmbH)
      A review. Mammalian homologs of the Drosophila melanogaster transient receptor potential (TRP) channels are the 2nd largest cation channel family within the superfamily of hexahelical cation channels. Most mammalian TRP channels function as homo-oligomers and mediate monovalent or divalent cation entry upon activation by a variety of stimuli. Because native TRP channels may be multimeric proteins of possibly complex compn., it has been difficult to compare cation conductances in native tissues to those of clearly defined homomeric TRP channel complexes in living cells. Therefore, the possibility of heteromeric TRP channel assembly has been investigated in recent years by several groups. As a major conclusion of these studies, most heteromeric TRP channel complexes appear to consist of subunit combinations only within relatively narrow confines of phylogenetic subfamilies. Although the general capability of heteromer formation between closely related TRP channel subunits is now clearly established, investigators are only beginning to understand whether these heteromeric complexes are of physiol. significance. Here, the author summarizes current knowledge on the promiscuity and specificity of the assembly of channel complexes composed of TRPC-, TRPV-, and TRPM-subunits of mammalian TRP channels.
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    TRPV-ARD structures used in this study (Table S1), structural similarity between TRPV4-ARD and other TRPV-ARDs (Table S2), molecular dynamics simulations of rat TRPV1-ARD (Table S3), Tm values of wild-type and mutant TRPV4-ARD proteins (Table S4), hTRPV4-ARD cysteine modification assay (Figure S1), stabilities of TRPV1-ARD in equilibrium and SMD simulations (Figure S2), and thermal stabilities of wild-type and mutant TRPV4-ARD proteins (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.


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