Volume-regulated anion channels (VRACs) formed by leucin-rich repeat containing 8 (LRRC8) proteins play a pivotal role in regulatory volume decrease by mediating the release of chloride and organic osmolytes. Apart from the regulation of cell volume, LRRC8/VRAC function underlies numerous physiological processes in vertebrate cells including membrane potential regulation, glutamate release and apoptosis. LRRC8/VRACs are also permeable to antibiotics and anti-cancer drugs, representing therefore important therapeutic targets. The activation mechanisms for LRRC8/VRACs are still unclear. Besides through osmotic cell swelling, LRRC8/VRACs can be activated by various stimuli under isovolumetric conditions. Sphingosine-1-phosphate (S1P), an important signalling lipid, which signals through a family of G protein-coupled receptors (GPCRs), has been reported to activate LRRC8/VRACs in several cell lines. Here, we measured inter-subunit Förster resonance energy transfer (FRET) and used whole-cell patch clamp electrophysiology to investigate S1P-induced LRRC8/VRAC activation. We systematically assessed the involvement of GPCRs and G protein-mediated signal transduction in channel activation. We found that S1P-induced channel activation is mediated by S1PR1 in HeLa cells. Following the downstream signalling pathway of S1PR1 and using toxin-mediated inhibition of the associated G proteins, we showed that Gβγ dimers rather than Gαi or Gαq play a critical role in S1P-induced VRAC activation. We could also show that S1P causes protein kinase D (PKD) phosphorylation, suggesting that Gβγ recruits phospholipase Cβ (PLCβ) with the consequent PKD activation by diacylglycerol. Notably, S1P did not activate LRRC8/VRAC in HEK293 cells, but overexpression of Gβγ-responsive PLCβ isoform could facilitate S1P-induced LRRC8/VRAC currents. We thus identified S1PR1-mediated Gβγ-PLCβ signalling as a key mechanism underlying isosmotic LRRC8/VRAC activation.
