Abstract
All vertebrate cells activate Cl– currents (ICl,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 ICl,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl,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 ICl,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl,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 ICl,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.
Highlights
Cell swelling activates Cl− currents (ICl,swell) that are known as volume-regulated anion channels (VRAC) (Jentsch, 2016; Pedersen et al, 2016; Strange et al, 2019)
Overexpressed LRRC8A/C was very rapidly activated by hypotonic bath solution (Hypo)-induced cell swelling (Figure 1B)
The cell line was ideal to examine the effects of overexpressed TMEM16A and LRRC8A/C
Summary
Cell swelling activates Cl− currents (ICl,swell) that are known as volume-regulated anion channels (VRAC) (Jentsch, 2016; Pedersen et al, 2016; Strange et al, 2019). VRAC is identified as LRRC8/SWELL1 (Qiu et al, 2014; Voss et al, 2014). The mechanism for activation of VRAC/LRRC8 channels remains incompletely understood. While lowering intracellular ionic strength is an essential prerequisite (Cannon et al, 1998; Sabirov et al, 2000; Syeda et al, 2016), intracellular Ca2+ was shown to be important for activation of ICl,swell and for cellular volume regulation (Mccarty and O’neil, 1992; Lemonnier et al, 2002; Akita et al, 2011; Benedetto et al, 2016; Liu et al, 2019)
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