Direct or indirect regulation of calcium-activated chloride channel by calcium

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Despite their physiological importance, the molecular identity of CaCCs has not been fully investigated until now. Recently, transmembrane 16A (TMEM16A) was demonstrated by three independent research groups to be a strong candidate for the CaCC molecular basis. To further investigate the electrophysiological characteristics, we constructed TMEM16A (abcd) stably transfected HEK293 cell lines and carried out whole-cell and excised inside-out patch-clamp experiments. The TMEM16A channel was Ca(2+)-dependent in both patch configurations. The TMEM16A current could be strongly inhibited by niflumic acid, and when Cl(-) was substituted by gluconate ions, the current was reduced considerably. In inside-out configuration, TMEM16A channel was time-independent but voltage-dependent, in which the half-maximum activating free Ca(2+) concentration was 63 nM at 80 mV. While in whole-cell configuration, the current was both time- and voltage-dependent. About the rectification feature, the TMEM16A current also showed distinct characteristics in the two patch configurations. In whole cells, the TMEM16A channel expressed outward rectification at low Ca(2+) concentration but when the Ca(2+) concentration was high it became linear. On the contrary, in inside-out configuration, it always expressed outward rectification. Comparing the different characteristics in the two configurations, some underlying mechanisms remain to be identified, which is discussed with respect to direct or indirect activation. There was irreversible rundown in this channel.