Fig. 4: TRPV4–RhoA interactions result in reciprocal functional inhibition.
From: Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension
a MN-1 cells transfected with WT or R237L TRPV4-GFP alone or in combination with WT RhoA or constitutively active RhoA (Q63L) were loaded with calcium indicator Fura-2 AM and treated with hypotonic saline (30 mM NaCl) at time 0. WT RhoA inhibits TRPV4-mediated calcium influx of WT TRPV4 but not R237L TRPV4. RhoA-Q63L does not inhibit WT TRPV4-mediated calcium influx. Scale bar, 50 µm. b Change in Fura ratio (340/380) divided by baseline Fura ratio (ΔF/F) in experiments shown in a. n = 9 coverslips per condition. c Change in Fura ratio (340/380) divided by baseline Fura ratio at time = 250 s in experiments shown in a. One-way ANOVA with Tukey post hoc test, n = 9 coverslips per condition, ***p = 0.0002. d T-Rex-TRPV4 cells were uninduced or induced with tetracycline (15 ng/ml, 16 h), then treated with vehicle or RhoA stimulator colchicine (10 µg/ml, 45 min) followed by analysis of RhoA activation. WT TRPV4 expression reduces basal RhoA activation (lane 2 vs. 1) and colchicine-stimulates RhoA activation (lane 4 vs. 3), whereas neuropathy mutant TRPV4 fails to influence basal or stimulated RhoA activity. e Densitometry of band intensities (active RhoA/total RhoA); representative blots shown in d. Data for T-Rex-TRPV4WT and T-Rex-TRPV4R269C conditions were normalized to their respective uninduced and vehicle-treated condition from each cell line. WT TRPV4 but not neuropathy mutant TRPV4 inhibits RhoA activation. One-way ANOVA with Tukey post hoc test, n = 6, *p = 0.046, ***p = 0.0004, #p = 0.030. f HEK293T cells were transfected with empty vector, GFP, p63RhoGEF-mVenus, or p63RhoGEF-mVenus and WT TRPV4-FLAG in the presence of HC067 (0.5 µM), and RhoA activity was analyzed after 24 h. Expression of p63RhoGEF leads to increased RhoA activation that is inhibited by WT TRPV4. g Densitometry of active RhoA band intensity, normalized to empty vector transfection; representative blot shown in f. One-way ANOVA with Tukey post hoc test, n = 7, *p = 0.028, #p = 0.020. Data are presented as means ± SEM. Further details regarding the number of times experiments were repeated are presented in “Methods” under “Statistics and reproducibility”.
