Ca Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl Channels.

All vertebrate cells activate Cl currents (I ) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca-activated Cl channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca release from the endoplasmic reticulum and to cause subsequent Ca influx. It is suggested that TMEM16A/F support intracellular Ca signaling and thus Ca-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to I . In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of I by hypotonic bath solution (Hypo; 200 mosm/l) was Ca dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca concentrations. In HT cells, knockdown of endogenous TMEM16A attenuated I and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of I by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IPR; RyR), as well as by inhibiting Ca influx. The data suggest that TMEM16A contributes directly to I as it is activated through swelling-induced Ca increase. As activation of VRAC is shown to be Ca-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca increase in submembraneous signaling compartments by means of ER tethering.