Supplementary Figure 2: Ion permeation and blockage of Xenopus TRPV4 and TRPV4_cryst.

a, Confocal images of CosM6 cells expressing full-length wild type Xenopus TRPV4 fused with EGFP (left panel) and the crystal construct TRPV4_cryst fused with EGFP (right panel). TRPV4_cryst shows altered expression pattern and is mainly localized to intracellular organelles. b, K⁺ and Cs⁺ conduction of wild type Xenopus TRPV4 and TRPV4_cryst. Single-channel current-voltage dependence of TRPV4 in symmetrical 150 mM KCl (open circles, n = 8), in symmetrical 150 mM CsCl (open squares, n = 7), and TRPV4_cryst in symmetrical 150 mM KCl (filled circles, n = 12), in symmetrical 150 mM CsCl (filled squares, n = 6). Currents were measured in inside-out excised patches upon application of GSK101 (10 nM for wild type TRPV4, 20 nM to 5 μM in KCl and 5 μM in CsCl for TRPV4_cryst) from the cytoplasmic side. Wild type TRPV4 has a unitary conductance of 280 ± 11 pS and 191 ± 8 pS at −100 mV membrane potential in symmetrical 150 mM KCl and symmetrical 150 mM CsCl respectively. Under the same conditions, TRPV4_cryst has a unitary conductance of 272 ± 13 pS and 185 ± 9 pS in symmetrical 150 mM KCl and symmetrical 150 mM CsCl respectively. c, Representative traces of activation of TRPV4_cryst by GSK101 in symmetrical 150 mM KCl (top panel, 20 nM GSK101) and in 150 mM CsCl (bottom panel, 5 μM GSK101). Membrane potential is −120 mV. d, Ca²⁺ and Ba²⁺ permeation of wild type Xenopus TRPV4. Inside-out membrane patches were excised in symmetrical BaCl₂ (75 mM) or CaCl₂ (75 mM) buffers. Wild type TRPV4 currents (Ba²⁺, black; Ca²⁺, grey) were induced by perfusion of the bath (cytoplasmic side) with K_int containing 10 nM GSK101. Reversal potentials are 27 mV for BaCl₂/KCl and 33 mV for CaCl₂/KCl, respectively. After liquid junction potential correction, the corresponding permeability ratios were calculated to be P_Ba/P_K = 3.4 (n = 8) and P_Ca/P_K = 4.8 (n = 5). Basal currents in the absence of GSK101 were very low and only noticeable at extreme potentials (−100 and 100 mV). e, Gd³⁺ blockage of TRPV4_cryst. GSK101 activation (1 μM) of TRPV4_cryst (filled squares, n = 3, mean ± SEM) in CosM6 cells was assessed by relative efflux of ⁸⁶Rb⁺. Basal ⁸⁶Rb⁺ efflux was measured in cells without GSK101 stimulation (empty circles, n = 3, mean ± SEM). GSK101-induced ⁸⁶Rb⁺ efflux was inhibited in the presence of 1 mM GdCl₃ (empty squares, n = 3, mean ± SEM). f, GSK2193874 (GSK219) inhibition of Xenopus TRPV4 and TRPV4_cryst. GSK101 activation (40 nM) of wild type TRPV4 (black diamonds, n = 3, mean ± SEM) and TRPV4_cryst (black squares, n = 3, mean ± SEM) in CosM6 cells was assessed by relative efflux of ⁸⁶Rb⁺. Basal ⁸⁶Rb⁺ efflux was measured in cells transfected with empty vector and treated with GSK101 (40 nM) (black circles, n = 3, mean ± SEM). GSK219 (10 μM) partially inhibits GSK101-induced ⁸⁶Rb⁺ effluxes for both wild type Xenopus TRPV4 (grey diamonds, n = 3, mean ± SEM) and TRPV4_cryst (grey squares, n = 3, mean ± SEM).