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

Clinical findings.

We examined 4 individuals from 2 families (figure 1B) exhibiting features consistent with the CMT2C phenotype,^10,23 including distal limb weakness, hoarseness of voice, and vocal fold paresis (table). Three of the 4 individuals underwent nerve conduction studies, which revealed axonal neuropathy. Laryngoscopy in 2 patients confirmed vocal focal paresis. In individual IV.1 of family 2, laryngoscopy revealed bilateral vocal fold paresis with vocal fold movement at 15%–20% of normal. Vocal fold EMG in this individual confirmed a reduction in motor unit number, consistent with chronic denervation. None of the participants exhibited evidence of skeletal abnormalities. Family 2 has a 3-generation history of limb weakness and hoarseness on the maternal side (figure 1B).

Detection of novel mutations in TRPV4.

Genetic testing of family 1 revealed a heterozygous missense mutation (c.709C>G) in exon 4 of TRPV4 (RefSeq accession number NM_021625.4) in both examined individuals. This nucleotide change results in an arginine to glycine substitution at amino acid 237 (p.Arg237Gly) of TRPV4. A heterozygous sequence change (c.710G>T) affecting amino acid 237 of TRPV4 was also detected in the 2 examined individuals in family 2, resulting in an arginine to leucine substitution (p.Arg237Leu) at this residue. Neither nucleotide substitution is present in the NHLBI Exome Sequencing Project Exome Variant Server, the 1000 Genomes Project, Exome Aggregation Consortium, or dbSNP. Like most known neuropathy-causing TRPV4 mutations, Arg237 is located in the TRPV4-ARD (third ankyrin repeat; figure 1A) and is highly conserved across vertebrate species (figure 1C). In silico analyses predict both mutations to be deleterious (PolyPhen-2 HumVar scores: p.Arg237Gly, 0.785; p.Arg237Leu, 0.713) and disease-causing (MutationTaster scores: p.Arg237Gly, 1; p.ArgR237Leu, >0.999).

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-ARD¹⁷ 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.

Figure 2 Arg237 forms part of a neuropathy-causing mutation cluster, distinct from clusters of skeletal dysplasia–causing mutations

In all panels, Arg237 is represented in magenta; residues mutated in neuropathy, skeletal dysplasia, and osteoarthropathy are shown in blue (R232, R269, R315, R316), yellow (E183, K197, L199, Q239, E278, T295, I331, D333, V342, K407, F471, L523, Y591, F592, R594, L596, G600, Y602, I604, R616, F617, L618, M625, L709, A716), and orange (G270, R271, F273), respectively. Mutated residues reported as causing mixed nerve/bone disease are shown in green (A217, K276, E278, S542, V620, T740). (A) Ribbon diagram of the TRPV4-ARD (residues 148–392) with disease-causing mutation positions marked with spheres. Arg237 is located on the convex face of the ankyrin repeat domain (ARD) within the previously identified cluster of arginine residues (shown as sticks) mutated in hereditary neuropathy. (B) Homology model of human TRPV4 based on the TRPV1 structure.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 stature⁶ 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 (C) Top-down view of the ion channel (as seen from the extracellular side) in surface representation. The transmembrane domain is colored cyan and the cytosolic regions gray. The cluster of neuropathy-causing mutations (indicated in 1 subunit by the arrow) is oriented toward the intracellular leaflet of the plasma membrane. (D) Expanded view of the region indicated in panel B showing 1 subunit in cyan ribbon representation and the neighboring subunit in surface representation. Several skeletal dysplasia–causing mutations occur at this intersubunit interface.

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 channel²⁰ 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 β-sheet structure arising from the ARD-to-transmembrane 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 β-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 and the mutant channels, including p.Ile331Phe and p.Arg271Pro (figure 3B). To examine effects on channel function, we performed ratiometric calcium imaging to measure cytosolic calcium levels ([Ca²⁺]i) of transiently transfected MN-1 (a motor neuron–like cell line) and HEK293T cells 24 hours after transfection. Expression of both p.Arg237Gly and p.Arg237Leu resulted in significant elevations of basal [Ca²⁺]i levels relative to the WT channel in both cell types, of comparable magnitude to known disease-causing TRPV4 mutants (figure 3, C and D). HEK293T cells expressing p.Arg237Gly and p.Arg237Leu also exhibited significantly increased levels of cytotoxicity relative to TRPV4^WT-transfected cells (figure 3E). Both the effects on [Ca²⁺]i and cell viability were abrogated by application of HC-067047 (figure 3, C–E), consistent with a mutation-dependent increase in channel activity.^4,5,7,28 It is interesting that despite causing significant elevations of basal [Ca²⁺]i, expression of the osteoarthropathy-causing mutant p.Arg271Pro was not associated with cytotoxicity (figure 3E). To confirm that the elevations in [Ca²⁺]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.

Figure 3 p.Arg237Gly and p.Arg237Leu mutations do not alter the subcellular localization of TRPV4 but cause elevations of basal [Ca²⁺]i and significant cytotoxicity

(A, B) Representative images of HEK293T cells transfected with wild-type (WT) or mutant TRPV4-FLAG and labeled immunocytochemically for FLAG (red) to reveal the subcellular distribution of TRPV4. Cells were costained for the transferrin receptor (green) and with DAPI (blue) to delineate individual cells and nuclei, respectively. These studies included examination of known neuropathy–causing (p.Arg269Cys), skeletal dysplasia–causing (p.Ile331Phe), and osteoarthropathy-causing (p.Arg271Pro) TRPV4 mutations. Transfected cells were cultured in the absence (A) or presence (B) of the TRPV4 antagonist HC-067047 (5 μM). All images are shown at the same magnification, and the scale bar represents 20 μm. (C, D) Quantification of baseline Fura-2 ratios in transiently transfected MN-1 (C) and HEK293T (D) cells reveals significant increases in basal [Ca²⁺]_i with expression of each mutant channel that are blocked by the TRPV4 antagonist HC-067047 (5 μM). (E) Quantification of cell death in HEK293T cells 24 hours after transfection using an LDH cytotoxicity assay. Cells expressing TRPV4 mutants, with the exception of p.Arg271Pro, show significantly increased levels of cytotoxicity relative to TRPV4^WT-expressing cells; this increase is abrogated by HC-067047 (5 μM). **p < 0.001, ***p < 0.0001. Data in C and D are averaged from >100 transfected cells, while data in E are averaged from 3 independent transfections. Error bars, SEM. (F, G) Immunoblotting of whole lysates from HEK293T cells 24 hours after transfection incubated in the absence (F) or presence (G) of HC-067047 (5 μM).