Ca²⁺ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl^- Channels

All vertebrate cells activate Cl^- currents (I_{Cl ,swell}) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca²⁺-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 Ca²⁺ release from the endoplasmic reticulum and to cause subsequent Ca²⁺ influx. It is suggested that TMEM16A/F support intracellular Ca²⁺ signaling and thus Ca²⁺-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to I_{Cl ,swell}. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of I_{Cl ,swell} by hypotonic bath solution (Hypo; 200 mosm/l) was Ca²⁺ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca²⁺ concentrations. In HT₂₉ cells, knockdown of endogenous TMEM16A attenuated I_{Cl ,swell} and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of I_{Cl ,swell} by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP₃R; RyR), as well as by inhibiting Ca²⁺ influx. The data suggest that TMEM16A contributes directly to I_{Cl ,swell} as it is activated through swelling-induced Ca²⁺ increase. As activation of VRAC is shown to be Ca²⁺-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca²⁺ increase in submembraneous signaling compartments by means of ER tethering.