Fig. 3

Quantitative relationships between endogenous PIP₂ level and whole-cell TRPM4 current amplitude for WT and E7K mutant. (A) Graded depletion of endogenous PI(4,5)P₂ was attained by applying a set of depolarizing pulses of incremental duration (300–2000 ms; from −60 to 120 mV). To minimize time-dependent changes due to desensitization, a relatively low concentration of Ca²⁺ (1 μM) was included in the pipette to induce TRPM4 currents under the whole-cell conditions. The tree panels in the figure denote the simultaneously recorded whole-cell TRPM4 current (I_m) and concomitant FRET ratio (FR) in response to depolarizing pulses of incremental duration (300–2000 ms; V_m). Prolongation of depolarizing pulses resulted in the progressive inhibition of whole-cell TRPM4 currents, the extent of which was greater for WT than E7K, despite the same degree of FR decrease (i.e., PIP₂ depletion). (B) Duration-dependent inhibition of the whole-cell TRPM4 current by depolarization-induced PIP₂ depletion for WT and E7K (open vs. filled circles). The duration-dependent inhibition is defined by using the ratio of TRPM4 current amplitudes before (I_pre) and after (I_post) each depolarization according to the formula: fractional inhibition = 1 − I_post/I_pre, which is plotted against the pulse duration. FR change is also displayed together (dashed curve). (C) The relationships between endogenous PIP₂ concentration and WT- or E7K-TRPM4 channel activity (normalized whole-cell current amplitude). Endogenous PIP₂ concentration was estimated from the FR value after the previous study [16] according to the formula: FRFRmax≈F2={1/(1+Kd[PI(4,5)P2])}², where FR_max was determined from PIP5K-overexpressing cells as a 1.2-fold higher value than that evaluated from the control cells [16]. The apparent K_d values of PI(4,5)P₂ binding to WT- TRPM4 channels and E7K-mutant TRPM4 channels determined by this method were 1.06 ± 0.05 μM and 0.97 ± 0.02 μM, respectively. *: p < 0.05 with ANOVA followed by Tukey’s post hoc tests (n = 5). (D) Time courses of the whole-cell TRPM4 currents recovering from depolarization-induced, DrVSP-mediated inhibition for WT (red curve in a) and E7K (blue curve in a). The time constant of recovery (τ) was determined by mono-exponential fitting, which resulted in values of 17.56 ± 5.29 and 13.71 ± 4.24 s for WT and E7K, respectively (b). *: p < 0.05 with unpaired t-test, respectively (n = 5).