Article Figures & Data
Figures
- Figure 1 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 = unaffected, black = affected, gray = unclear disease status, *DNA collected). (C) The Arg237 residue of TRPV4 has been highly conserved through vertebrate evolution. ARD = ankyrin repeat 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.
- 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 TRPV4WT-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).
Tables
Letters: Rapid online correspondence
NOTE: All contributors' disclosures must be entered and current in our database before comments can be posted. Enter and update disclosures at http://submit.ng.neurology.org. Exception: replies to comments concerning an article you originally authored do not require updated disclosures.
- Stay timely. Submit only on articles published within the last 8 weeks.
- Do not be redundant. Read any comments already posted on the article prior to submission.
- 200 words maximum.
- 5 references maximum. Reference 1 must be the article on which you are commenting.
- 5 authors maximum. Exception: replies can include all original authors of the article.
- Submitted comments are subject to editing and editor review prior to posting.