Fig. 9.
Mutation of serine 528 to alanine attenuates TMEM16A rundown to a level that is quantitatively similar to that exerted by specific calmodulin-dependent protein kinase II (CaMKII) inhibitors. A: graph showing the mean time courses of changes of normalized late Ca2+-activated Cl− currents (IClCa) elicited by depolarizing steps to +90 mV applied at a frequency of 0.1 Hz from holding potential of −50 mV for wild-type (WT) TMEM16A (closed circles) or TMEM16A T528A mutant in the absence (open circles) or presence of 10 μM KN-93 in the pipette solution (open squares). All currents were normalized to the initial current recorded immediately upon cell membrane rupture. The S528A mutation significantly reduced the rundown of TMEM16A-induced IClCa HEK-293 cells over the course of a 10-min period of cell dialysis with 5 mM ATP (S528A: 0.589 ± 0.087, n = 19; S528A + KN-93: 0.602 ± 0.088, n = 9; WT: 0.327 ± 0.030, n = 23; P < 0.01). B, left: mean bar graph showing the magnitude of the initial late TMEM16A current density ± SE (in pA/pF) recorded after seal rupture (t = 0) in cells dialyzed with 5 mM ATP for wild-type TMEM16A (closed bar; n = 14) and the S528A mutant (open bar; n = 8). B, right: mean current-voltage relationships for late IClCa recorded from wild-type TMEM16A (closed circles) and the S528A mutant after seal rupture (t = 0). Currents in each group were normalized to that recorded at +120 mV in their respective group. Data points in each group are means ± SE. C: the time course plots shown in Fig. 4, A and C, were superimposed to those of A to illustrate the similarity between the attenuation of rundown of TMEM16A produced by the S528A mutation and that elicited by the 2 CaMKII inhibitors autocamtide-2-related inhibitory peptide (ARIP) and KN-93. One-way ANOVA analysis revealed no significant difference between the 3 groups of data after 10 min of cell dialysis with 5 mM ATP (P > 0.05). D: graph showing the mean time courses of changes of normalized late IClCa elicited by depolarizing steps to +90 mV applied at a frequency of 0.1 Hz from holding potential of −50 mV for WT TMEM16A (closed triangles) or TMEM16A S528A mutant (open triangles) dialyzed with 0 ATP. The TMEM16A S528A mutant decayed over a 10 min period to a level that was ~16% of the initial amplitude, which was not significantly different from WT TMEM16A currents (1.038 ± 0.098, n = 13; P = 0.13). For all panels: †P < 0.01; ††P < 0.001; n.s., not significant.