Fig. 4

Fig. 4 Different voltage dependence between the WT-TRPM4 channel and E7K-mutant TRPM4 channel after VSP-induced PIP2 depletion. (A) The left and right three panels show data from WT and E7K, respectively. Top panel (V_m): a dual-ramp protocol consisting of an ascending ramp of 1 s, sustained depolarization at +120 mV for 2 s, and a descending ramp of 1 s. The ramps spanned between −100 and 200 mV. Middle and bottom panels (I_m and FR): representative simultaneous recordings of membrane currents and FRET ratios (which represent the endogenous PIP₂ level) during V_m changes. (B) Chord conductance (G)-V_m relationships for WT- and E7K-mutant TRPM4 channels before (open circles) and after (filled circles) DrVSP-mediated PIP2 depletion. The values of G/Gmax were calculated from the data as shown in A, and the G_max was taken as the maximal G value at 200 mV of the ascending ramp. After PIP₂ depletion, the voltage dependence of the WT-TRPM4 channel was remarkably rightward shifted. In contrast, this shift was only marginal for E7K mutant, especially around the resting membrane potential. After fitting of these G-V data to the Boltzmann equation: G/G_max = G₀/G_max + (G_max − G₀)/(1 + exp((V_0.5 − V_m)/s) (V_m, V_0.5, s: membrane potential, half-maximal activation voltage, slope factor, respectively), the V0.5 values before and after DrVSP activation: 62.07 ± 7.32 and 83.31 ± 9.53 vs. 59.63 ± 10.79 and 219.84 ± 75.37; and the s values before and after DrVSP activation (mV): 58.79 ± 11.46 mV and 70.45 ± 12.69 mV vs. 60.04 ± 18.52 mV and 101.58 ± 26.29, were obtained for the E7K-mutant and WT-TRPM4 channels, respectively (n = 8). Note that in both WT and E7K, there appeared to be a substantial non-voltage-dependent component (G₀/G_max) at very negative potentials, which may reflect incomplete PIP₂ depletion by a 2-s depolarization to +120 mV.