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Novel mutations highlight the key role of the ankyrin repeat domain in TRPV4-mediated neuropathy

Objective: To characterize 2 novel TRPV4 mutations in 2 unrelated families exhibiting the Charcot-Marie-Tooth disease type 2C (CMT2C) phenotype. Methods: Direct CMT gene testing was performed on 2 unrelated families with CMT2C. A 4-fold symmetric tetramer model of human TRPV4 was generated to map the locations of novel TRPV4 mutations in these families relative to previously identified disease-causing mutations (neuropathy, skeletal dysplasia, and osteoarthropathy). Effects of the mutations on TRPV4 expression, localization, and channel activity were determined by immunocytochemical, immunoblotting, Ca2+ imaging, and cytotoxicity assays. Results: Previous studies suggest that neuropathy-causing mutations occur primarily at arginine residues on the convex face of the TRPV4 ankyrin repeat domain (ARD). Further highlighting the key role of this domain in TRPV4-mediated hereditary neuropathy, we report 2 novel heterozygous missense mutations in the TRPV4-ARD convex face (p.Arg237Gly and p.Arg237Leu). Generation of a model of the TRPV4 homotetramer revealed that while ARD residues mutated in neuropathy (including Arg237) are likely accessible for intermolecular interactions, skeletal dysplasia–causing TRPV4 mutations occur at sites suggesting disruption of intramolecular and/or intersubunit interactions. Like previously described neuropathy-causing mutations, the p.Arg237Gly and p.Arg237Leu substitutions do not alter TRPV4 subcellular localization in transfected cells but cause elevations of cytosolic Ca2+ levels and marked cytotoxicity. Conclusions: These findings expand the number of ARD residues mutated in TRPV4-mediated neuropathy, providing further evidence of the central importance of this domain to TRPV4 function in peripheral nerve.

Results: Previous studies suggest that neuropathy-causing mutations occur primarily at arginine residues on the convex face of the TRPV4 ankyrin repeat domain (ARD). Further highlighting the key role of this domain in TRPV4-mediated hereditary neuropathy, we report 2 novel heterozygous missense mutations in the TRPV4-ARD convex face (p.Arg237Gly and p.Arg237Leu). Generation of a model of the TRPV4 homotetramer revealed that while ARD residues mutated in neuropathy (including Arg237) are likely accessible for intermolecular interactions, skeletal dysplasia-causing TRPV4 mutations occur at sites suggesting disruption of intramolecular and/or intersubunit interactions. Like previously described neuropathy-causing mutations, the p.Arg237Gly and p.Arg237Leu substitutions do not alter TRPV4 subcellular localization in transfected cells but cause elevations of cytosolic Ca 21 levels and marked cytotoxicity. Mutations in the transient receptor potential vanilloid 4 gene (TRPV4), encoding a Ca 21permeable cation channel, result in 3 autosomal dominant diseases of peripheral nerve: Charcot-Marie-Tooth disease type 2C (CMT2C), congenital distal spinal muscular atrophy, and scapuloperoneal spinal muscular atrophy. [1][2][3][4][5][6][7][8][9] While these diseases are clinically heterogeneous, a majority of patients have weakness of limb and vocal fold muscles. 10,11 In addition to hereditary neuropathy, dominant mutations in TRPV4 are associated with forms of skeletal dysplasia and osteoarthropathy. 12 TRPV4 mutations have also been described in individuals manifesting both skeletal dysplasia and either peripheral neuropathy or fetal akinesia. 13,14 Our understanding of how TRPV4 mutations result in such diverse disease phenotypes is currently limited, although several in vitro studies suggest that neuropathy-and skeletal dysplasia-causing mutants exhibit normal expression levels and localization but increased channel activity. 12 TRPV4 functions primarily as a homotetrameric channel expressed at the plasma membrane. 15 The cytoplasmic N-terminus of each protomer (figure 1A) contains a prominent ankyrin repeat domain (ARD) comprising 6 ankyrin repeats, a motif mediating proteinprotein/protein-ligand interactions. 16 Structural analyses indicate that neuropathy-causing TRPV4 mutations occur primarily at arginine residues clustered on the ARD convex face ( figure 1A). 4,5,17 In contrast, mutations associated with skeletal dysplasia occur throughout the protein, with the exception of the ARD convex face. 12 Osteoarthropathy-causing mutations reside within the third finger loop of the ARD. 18 In this article, we report 2 novel TRPV4 mutations in 2 families exhibiting the CMT2C phenotype. Both mutations occur at an arginine residue in the ARD (Arg237) not previously linked to peripheral neuropathy.

METHODS Participants and molecular genetic analyses.
Participants were evaluated at Stanford University Medical Center and the University of Washington Medical School. Weakness was graded as mild if the Medical Research Council scale score was $4/5, moderate if $3 and ,4, and severe if #2. Sensory loss was determined to be moderate or mild by the examiner based on vibration testing. Genomic DNA was isolated from blood leukocytes using standard extraction protocols and examined by direct CMT gene testing.
Homology model generation. The ARD is currently the only TRPV4 domain for which a high-resolution structure has been determined. 5,17 In this study, SWISS-MODEL 19 was used to generate a 4-fold symmetric tetramer model of human TRPV4 (residues 148-755 of 871) using the apo rat TRPV1 electron cryomicroscopy structure as a template (PDBID 3J5P 20 ). Rat TRPV1-ARD and human TRPV4-ARD have 56% sequence identity, and their core Ca atoms have a root mean square Two novel CMT2C-causing mutations identified at a highly conserved arginine residue in the TRPV4-ARD (A) Schematic illustrating the principal TRPV4 protein domains and the locations of neuropathy-causing mutations. *Novel mutations identified in the present study in 2 families with the Charcot-Marie-Tooth disease type 2C (CMT2C) phenotype. †A mutation found in a family with CMT2C and pronounced short stature. 6 ‡A mutation described in a single patient with CMT2 and skeletal dysplasia, 3 as well as in a single patient with axonal neuropathy and both the p.Val620Ile mutation and a second p.Arg151Trp TRPV4 variant. 37 (B) Pedigrees of families 1 and 2 demonstrating affected individuals (white 5 unaffected, black 5 affected, gray 5 unclear disease status, *DNA collected). (C) The Arg237 residue of TRPV4 has been highly conserved through vertebrate evolution. ARD 5 ankyrin repeat domain.
deviation of 1.6 Å, allowing us to place the experimentally determined TRPV4-ARD crystal structure with high confidence within our homology model. Therefore, after generation of a full model, the SWISS-MODEL-generated ARD was removed and replaced with the experimentally determined x-ray crystal structure of the human TRPV4-ARD (PDBID 4DX2, chain B, residues 148-389 17 ) after alignment to residues 350-389 (ankyrin repeat 6) in Coot. 21 To relieve any resulting clashes, 10 rounds of geometry minimization of residues 390-470 and 635-666 were performed in phenix.refine 22 with the 4-fold symmetry restrained. The C-terminal b-strand (residues 752-762 in the 3J5P TRPV1 structure) was then added to the homology model to illustrate the position of this structural feature. PyMOL was used for model analysis and image generation.
Immunocytochemistry. HEK293T figure 1A) and is highly conserved across vertebrate species (figure 1C). In silico analyses predict Structural analyses localize Arg237 to the convex face of the ARD. Ankyrin repeats comprise inner and outer helices followed by a connecting finger loop, and the 6 repeats of the TRPV4-ARD together form a hand-shaped domain with a concave palm surface and a convex surface analogous to the back of the hand. 16,17 Analysis of the high-resolution crystal structure of the human TRPV4-ARD 17 reveals that Arg237 is situated within the second finger loop, near 2 other arginine residues mutated in CMT2C (Arg232 and Arg269; figure 2A). Like other neuropathy-causing mutations in the TRPV4-ARD, Arg237 is situated on the convex face of the domain.  20 Known disease-causing mutation positions marked with spheres. One subunit of the TRPV4 tetramer is shown in cyan with the remainder shown in gray. The plasma membrane is denoted with black lines, and the arrow points to a cluster of skeletal dysplasia-causing mutations near the lower channel gate. As in figure 1, †indicates a mutation found in a family with Charcot-Marie-Tooth disease type 2C and pronounced short stature 6 and ‡indicates a mutation described in a single patient with CMT2 and skeletal dysplasia, 3 as well as in a single patient with axonal neuropathy and both the p.Val620Ile mutation and a second p.Arg151Trp TRPV4 variant. 37  The positioning of the ARD relative to the rest of the assembled channel and the plasma membrane has not been previously investigated. Like other TRPV channels, functional TRPV4 channels are principally homotetramers. 24 We used the recently described electron cryomicroscopy structure of the TRPV1 ion channel 20 to generate a model of the human TRPV4 homotetramer (figure 2B) that incorporates the experimentally determined TRPV4-ARD. An overall high sequence identity between TRPV1 and TRPV4 (;45%) allows us to map the positions of most disease-causing TRPV4 mutations in other regions onto this model with high confidence. 25 Several interesting observations arise from this mapping. First, the convex face of the ARD, on which neuropathy-causing mutations cluster, is on the outer surface of the TRPV4 tetramer, facing toward the cytosol at an angle of ;45°relative to the plasma membrane (figure 2, B and C). This orientation suggests that the ARD convex face would likely be readily accessible for protein-protein/protein-ligand interactions. Second, skeletal dysplasia-causing mutations form 2 main clusters within the channel (figure 2, B and D). The larger of these clusters is situated within the bottom half of the transmembrane region near the lower channel gate, consistent with the finding that many of these mutations increase constitutive channel activity. 26 The second cluster occurs on the concave surface of the ARD. In the tetrameric channel, this surface participates in intersubunit interactions connecting the ARD of one protomer to a small b-sheet structure arising from the ARD-totransmembrane domain linker and the C-terminus of the adjacent protomer ( figure 2D). The location of this second cluster suggests that disruption of this intersubunit interface may also increase constitutive channel activity. Third, osteoarthropathy-causing mutants are clustered in the third finger loop of the ARD, 18 which interacts with the b-sheet structure on one side but is exposed to the solvent on the other. Although no clear conclusion can be drawn from our model, this loop can be flexible, 17 and it is plausible that the mutations affect interactions with a regulator or trafficking chaperone that results in reduced cell surface localization, as previously observed. 18 No clear localization patterns were evident for mutations thought to cause both nerve and bone disease (green residues, figure 2 13,14 ).
p.Arg237Gly and p.Arg237Leu mutations alter TRPV4 function. To gain insights into the functional consequences of the p.Arg237Gly and p.Arg237Leu amino acid changes, we performed a series of in vitro studies examining the effects of heterologous expression of these mutant channels. To assess the magnitude of any observed effects, experiments were performed in parallel with known disease-causing TRPV4 mutants (neuropathy, p.Arg269Cys; skeletal dysplasia, p.Ile331Phe; osteoarthropathy, p.Arg271Pro; the locations of these residues are shown in figure 2A). Immunocytochemical analyses revealed that each of the neuropathy-causing mutants examined exhibited a subcellular distribution comparable to that of the WT channel, localizing to both the cytoplasm and the plasma membrane ( figure 3A). In contrast, the skeletal dysplasia-and osteoarthropathy-causing mutants localized primarily to the cytoplasm (figure 3Avi). Incubation of transfected cells in a TRPV4 channel antagonist (HC-067047) increased plasma membrane expression of both TRPV4 WT , as described previously, 27 4,5,7,28 It is interesting that despite causing significant elevations of basal [Ca 21 ]i, expression of the osteoarthropathy-causing mutant p.Arg271Pro was not associated with cytotoxicity ( figure 3E). To confirm that the elevations in [Ca 21 ]i and cytotoxicity caused by the neuropathy-and skeletal dysplasia-causing mutants did not result from increased expression levels, we performed immunoblot analyses of whole-cell lysates 24 hours after transfection. These analyses revealed reduced expression of these mutants relative to TRPV4 WT (figure 3F), likely reflecting their increased cytotoxicity. Immunoblots performed on cells incubated in HC-067047 to abrogate toxicity, however, demonstrated equivalent protein expression levels across the WT and mutant channels ( figure 3G). Taken together, the above results suggest that the p.Arg237Gly and p.Arg237Leu mutants traffic normally to the plasma membrane but exhibit a gain of function in channel activity. DISCUSSION The structure of ARDs with a conserved hydrophobic core and variable exposed residues enables these domains to interact with binding partners (inter-and intramolecular) with high specificity and affinity. 16 While these properties enable ARDs to perform diverse cellular functions, mutations in the ARDs of a number of proteins are associated with disease, including autism spectrum disorder (SHANK3), melanoma (p16), keratoderma (KANK2), and bare lymphocyte syndrome (RFXANK). [29][30][31][32] To date, 8 of 12 neuropathycausing mutations in TRPV4 occur within the TRPV4-ARD ( figure 1A). The identification in the present study of 2 additional TRPV4-ARD mutations (p.Arg237Gly and p.Arg237Leu) in 2 families with the CMT2C phenotype further underscores the importance of this domain in TRPV4-mediated neuropathy. Like previously described neuropathy-causing mutations in the TRPV4-ARD, p.Arg237Gly and p.Arg237Leu occur at an exposed arginine residue on the convex face of the ARD. Six of the 10 exposed arginine residues present on the convex face of TRPV4-ARD have now been associated with neuropathy. This cluster of arginine residues is highly conserved across vertebrate species but is not characteristic of the ARDs of other TRPV channel family members. 1,[3][4][5]8 We previously determined experimentally that the neuropathy-causing mutations R232C, R269C, R269H, R315W, and R316C (all surface-exposed arginine mutations) do not substantially alter the fold or stability of the human TRPV4-ARD, which suggests that the functional consequence of these mutations may instead lie in interactions with binding partners. 17 While the concave surface of the TRPV4-ARD has been shown to bind ATP, calmodulin, 5,17,33 and PI(4,5)P 2 , 34 no interactors of the ARD convex face have yet been identified. By generating a model of the TRPV4 homotetramer, we can better understand the functional consequences of these mutations by understanding them in the context of a structural model of the full channel. In this study, we showed that the ARD convex face is directed toward the cytosol and would thus likely be readily accessible for protein-protein/protein-ligand interactions. Furthermore, the orientation of this surface toward the plasma membrane suggests that the ARD convex surface may interact principally with membraneassociated factors. As the TRPV1 electron cryomicroscopy structure 20 on which our model is based did not include regions N-terminal to the ARD, it remains unclear how neuropathy-causing mutations in N-terminal regions of TRPV4 (figure 1A) localize relative to the ARD convex face. It is possible that the N-terminal region of TRPV4 influences access to binding partners of the ARD, as demonstrated for autism-associated mutations in the postsynaptic scaffolding protein SHANK3. 35 It is also likely that the channel adopts other conformations not yet seen experimentally, but the relative orientation of the ARD in relation to the membrane is restrained by extensive interactions with other regions of the channel, suggesting that it is unlikely to be altered dramatically.
Generation of a model of the TRPV4 homotetramer also enabled the mapping of TRPV4 mutations associated with skeletal dysplasia and osteoarthropathy. Skeletal dysplasia-causing mutations were found to occur primarily within 2 clusters: a large cluster situated within the bottom half of the transmembrane region near the lower channel gate and a second cluster on the concave surface of the ARD that appears to participate in intersubunit interactions. Together, these findings suggest that skeletal dysplasia-causing mutations may influence TRPV4 channel activity by altering intramolecular interactions within the assembled channel. As described previously, 18 osteoarthropathy-causing mutations localize to the tip of the ARD third finger loop. In the tetrameric channel model, the corresponding residues are part of a potentially flexible loop and could be accessible to regulatory partners. In addition, a lack of any clear localization patterns for mutations thought to cause both nerve and bone disease (green residues, figure 2) indicates that factors not accounted for by this model play an important role in TRPV4 protein function.
Heterologous expression studies indicate that p.Arg237Gly and p.Arg237Leu exhibit a subcellular distribution equivalent to TRPV4 WT , suggesting that these mutations do not influence channel assembly or trafficking. Expression of these mutants, however, results in elevations of [Ca 21 ]i and significant cytotoxicity, both of which are abrogated by application of a TRPV4 antagonist, consistent with increased channel activity. 4,5,7,8,28 The magnitude of these effects is comparable to that of known disease-causing TRPV4 mutants. Together, these results suggest that both the p.Arg237Gly and p.Arg237Leu mutations confer a gain of function in channel activity. While heterologous expression of both skeletal dysplasia-and osteoarthropathy-causing mutants (p.Ile331Phe and p.Arg271Pro, respectively) also resulted in significant increases in basal [Ca 21 ]i, this was not associated with significant cytotoxicity in p.Arg271Pro-expressing cells. Similarly, expression of the osteoarthropathy mutant p.Phe273Leu in porcine chondrocytes results in elevations of [Ca 21 ]i but does not cause the channel activity-dependent increases in gene expression characteristic of skeletal dysplasia-causing mutants. 36 Together, these results suggest that factors in addition to, or independent of, increased Ca 21 influx may contribute to disease pathogenesis in some forms of TRPV4 channelopathy.
A clear understanding of how different mutations in TRPV4 engender the diverse disease phenotypes characteristic of patients is only slowly emerging. 10,12 Elucidation of the etiologies underlying the different forms of TRPV4 channelopathy is important because TRPV4 is expressed at the plasma membrane and channel antagonism could therefore represent a therapeutic strategy, where appropriate. The identification of 2 novel mutations in the TRPV4-ARD highlights the key role of this domain in TRPV4mediated neuropathy. Our findings suggest that molecular pathogenesis in these neuropathies may be driven by alterations in interactions of the ARD convex face with as-yet-unidentified binding partners. Identification of these interactors and characterization of their effects on channel function will be central to determining how mutations in TRPV4 precipitate peripheral axonal degeneration.

AUTHOR CONTRIBUTIONS
J.M. Sullivan: study conceptualization and design, acquisition of data, analysis and interpretation, and drafting and revision of manuscript. C.M. Zimanyi: acquisition of data, analysis and interpretation, and revision of manuscript. W. Aisenberg and B. Bears: acquisition of data and revision of manuscript. D.-H. Chen and J.W. Day: acquisition of clinical data and revision of manuscript. T.D. Bird: study conceptualization and revision of manuscript. C.E. Siskind: study conceptualization and revision of manuscript. R. Gaudet: study conceptualization and design, analysis and interpretation, and revision of manuscript. C.J. Sumner: primary investigator of the study, study conceptualization and design, analysis and interpretation, and drafting and revision of manuscript.

STUDY FUNDING
Supported by NIH grant R21NS08757902 to C.J.S., MDA grant