Identification of LRRC8/VRAC channel structural elements required for regulation by cell volume increase and intracellular ionic strength

VRACs mediate volume regulatory Cl- and organic solute efflux and are encoded by Lrrc8a-e genes. Native LRRC8/VRACs are heteromers with unknown stoichiometry comprising the essential LRRC8A subunit and another paralog. We utilize a chimeric protein approach to define LRRC8/VRAC structure-function relationships. Functional chimeric channels with physiologically relevant properties require the presence of the first intracellular loop (IL1) of LRRC8A and the first extracellular loop of another paralog. Replacement of the C-terminus of LRRC8C or LRRC8E with the C-terminus of LRRC8A (8A(C-term)) gives rise to robust plasma membrane anion channel activity. 8C-8A(C-term) and 8E-8A(C-term) chimeric channels exhibit greatly reduced sensitivity to reduced intracellular ionic strength (IS) and cannot be activated by hypotonicity-induced cell volume increases of up to 4-fold. However, both channels could be activated by rapid, stepwise volume increases of 2-2.5-fold induced by positive pressure applied to the patch pipette. Addition of the LRRC8A IL1 to both chimeras restored normal sensitivity to hypotonicity-induced volume increases and IS. A cryo-EM structure of a LRRC8 chimeric channel comprising LRRC8C with 25 amino acids unique to the LRRC8A IL1, 8C-8A(IL125), suggested a possible functional interaction between 8A(IL125) and leucine-rich repeat motif 5, LRR5. Deletion of 9 cytoplasm-facing LRR5 amino acids or substitution with alanine greatly reduced volume and IS sensitivity of 8C-8A(IL125) channels. Point mutations in the 8A(IL125) region identified two unique lysine residues required for channel regulation. We conclude that the LRRC8 C-terminus is 1) required for channel volume and IS sensing, 2) that channel regulation requires a functional interaction between the unique 8A(IL125) region and LRR5 and 3) that LRR5 and the 8A(IL125) region are key components of the LRRC8/VRAC volume and IS sensing machinery.