Figure 1
Kinetic analysis of PIP2 binding sites on TRPC6 channels. TRPC6 currents evoked by OAG (50 μM) were recorded in the whole-cell clamp mode from HEK293 cells co-transfected with enzyme-defective mutant DrVSPC302S (A) or wild-type DrVSP (B). Strong depolarizations (+ 100 mV, 700 ms) were applied every 15 s (protocol displayed above). The areas enclosed by the dashed boxes are enlarged in (C,D). The deactivation (decay, t1/2) and reactivation (recovery, τ) kinetics were analyzed by fitting the equations described in the “Materials and methods” section (C,D, blue lines). Current inhibition upon PIP2 depletion was evaluated as the ratio of the currents after and before the depolarization (Ipost/Ipre). Multistate gating diagrams are shown in the bottom. Channel deactivation and reactivation process by PIP2 involves four closed states, corresponding to each of the monomeric subunit in the tetrameric channel (C), with a single open state (O). (E) Kinetics of PIP2 depletion and replenishment upon the activation of DrVSP were measured with FRET.
