The intracellular subdomain of the volume-regulated anion channel subunit LRRC8A is a hotspot of channel activation

Volume-regulated anion channels (VRACs), formed by heteromers of LRRC8 subunits, mediate the transport of anions and organic osmolytes in numerous physiological processes. Although their gating mechanism remains poorly understood, conformational rearrangements of the cytosolic leucine-rich repeat domains (LRRDs) have been implicated as a key step towards activation. Here, we identify the intracellular subdomain (ISD) of the obligatory LRRC8A as a key element coupling LRRD motion to pore opening. Substitution of leucine 402 with tryptophan (L402W) within a hydrophobic pocket of the ISD rendered LRRC8A homomers constitutively open, independent of osmotic stimulation or pharmacological modulation. Mutation of the nearby tryptophan 168 to leucine (W168L) also enhanced channel activity whereas the combination of both mutations (W168L-L402W) partly attenuated the hyperactive phenotype of L402W. FRET measurements revealed increased LRRD flexibility of L402W, which was reduced in W168L-L402W or by coexpression with an inhibiting sybody. The cryo-EM structure of L402W showed pronounced ISD rearrangements and outward tilting of the LRRDs, while the double mutant W168L-L402W contains features explaining its intermediate phenotype. Together, these data identify the ISD as a regulatory hub that transmits conformational changes from the LRRDs to the transmembrane gate of VRAC.