Fig. 8.

Substitution of threonine 273 or serine 528 to a neutral alanine had no impact on the regulation of TMEM16A-induced Ca²⁺-activated Cl⁻ currents (I_ClCa). A: representative current traces recorded immediately (t = 0 min) and after 2 (t = 2 min) and 10 min (t = 10 min) following membrane rupture demonstrating the time-dependent rundown of I_ClCa recorded from HEK-293 cells overexpressed with wild-type (WT) TMEM16A (TMEM16A WT; top left), the T273A (TMEM16A T273A; top right) or the S730A mutant (TMEM16A S730A; bottom left), all recorded in the presence of 5 mM ATP in the pipette solution. The currents were evoked by 1 s steps to +90 mV from a holding potential of −50 mV; the inward tail current was elicited by a 1 s return step to −80 mV (bottom right). B: mean bar graph showing the amplitude of the initial current recorded at +90 mV at time = 0 from a holding potential of −50 mV (protocol identical to that shown in A, bottom right). Each bar is a mean ± SE current density in pA/pF. C: graph showing the mean time courses of changes of normalized late I_ClCa elicited by depolarizing steps to +90 mV applied at a frequency of 0.1 Hz for WT TMEM16A (closed circles) and the T273A (open squares) and S730A (open circles) mutants. Each data point is a means ± SE; n, number of cells. Mutation of the threonine at position 273 or serine at position 730 to an alanine did not alter the time course and magnitude of TMEM16A-mediated I_ClCa rundown. For B and C: *P < 0.05; ††P < 0.001; n.s., not significant.