Volume-regulated Cl^- current: contributions of distinct Cl^- channels and localized Ca²⁺ signals

The swelling-activated chloride current (I_Cl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of I_Cl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl^- channels, such as anoctamin 1 (ANO1), were also suggested to contribute to I_Cl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of I_Cl,swell; we also explored the role of intracellular Ca²⁺ in I_Cl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to I_Cl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca²⁺ transients, and these Ca²⁺ signals were required to activate both the LRRC8A- and ANO1-dependent components of I_Cl,swell. Both I_Cl,swell components required localized rather than global Ca²⁺ for activation. Interestingly, while intracellular Ca²⁺ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca²⁺ transients linked to the I_Cl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.