Volume-regulated Cl current: contributions of distinct Cl channels and localized Ca signals.

The swelling-activated chloride current () is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of 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 . In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ; we also explored the role of intracellular Ca in 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 , with LRRC8A being the major component. Cell swelling induced oscillatory Ca transients, and these Ca signals were required to activate both the LRRC8A- and ANO1-dependent components of . Both components required localized rather than global Ca for activation. Interestingly, while intracellular Ca was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca transients linked to the activation were mediated by the G protein-coupled receptor-independent PLC isoforms.