Fig. 2

TRPV4 M713 mutations in GCLJ are predicted to affect channel function and are associated with increased channel activity. a Schematic diagrams of the TRPV4 channel protein domains, including six transmembrane segments (S1–6), pore-forming region, ankyrin repeat domains (ANK1–6), proline rich domain (PRD), and calmodulin (CaM)-binding site. The position of each TRPV4 mutation detected in GCLJ is represented by a star or a triangle, along with the number of affected cases. b Model of TRPV4 protein in its homo-tetrameric closed state with the sphere representation of M713 residue within the transmembrane domain. c Closed state of TRPV1 (PDB ID:3J5P) and d open state of TRPV1 (PDB ID:5IRX), modeled with TRPV4 M713V. Surrounding hydrophobic residues are shown; residues are labeled using TRPV4 numbering. e Cell death assay on HEK293 cells expressing exogenous wild-type (WT) and mutant (M713I and M713V) TRPV4. TRPV4 mutant proteins in HEK293 cells lead to increased cell death (middle), which could be prevented by incubation with the ion channel blocker RuR (right). The percentage of cells in each quadrant is indicated as follows: lower left, live cells; lower right, early apoptosis, upper right, late apoptosis; upper left, necrosis. Representative data of three biological replicates are shown. f Representative traces of TRPV4 currents recorded in HEK293 cells before (constitutive activity) and after the application of TRPV4 agonist GSK1016790a (GSK101, 100 nM). Currents were recorded using the conventional whole-cell configuration and 300-ms voltage ramps (−100 to 100 mV, from a holding potential of −50 mV); ruthenium red (RuR, 1 µM) was included in the bath solution. Vertical scale bar, 100 pA/pF; horizontal scale bar, 100 ms. g Individual-value plots of outward current recorded at 100 mV in the absence of GSK101 (mean ± s.e.m, ^**P < 0.01, ^***P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test, WT, n = 13; M713V, n = 14; M713I, n = 13). h Individual-value plot of currents recorded at 100 mV from dialyzed HEK293 cells treated with 100 nM GSK101 and in the presence of 1 µM RuR (mean ± s.e.m, ^*P < 0.05, ^**P < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test, WT, n = 9; M713V, n = 15; M713I, n = 10). Black circles, WT; green squares, M713V; blue triangles, M713I