Oligomeric structure of LRRC8/VRAC channels: Arguments for a heptameric conformation

LRRC8 proteins form large-pore heteromeric volume regulated anion/organic solute channels, VRACs, comprising the LRRC8A subunit and another LRRC8 paralog. Heterologous expression of LRRC8A alone gives rise to homomeric channels with non-physiological properties. Purified LRRC8A protein forms narrow-pore homohexameric channels. Recent cryo-EM structures of purified LRRC8A+LRRC8C proteins revealed a narrow-pore heterohexameric structure with stoichiometries of 5A:1C and 4A:2C. We utilize a chimeric protein approach to define structure-function relationships of LRRC8/VRAC channels. A LRRC8 chimera comprising LRRC8C with 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A, 8C-8A(IL125), gives rise to homomeric channels with normal functional properties. Purified 8C-8A(IL125) protein forms a homoheptameric channel with a large pore diameter similar to that of native LRRC8/VRACs. DCPIB is a LRRC8/VRAC inhibitor. 8C-8A(IL125) homomeric and LRRC8A+LRRC8C heteromeric channels exhibit native DCPIB pharmacology. In contrast, the DCPIB pharmacology of LRRC8A homohexameric channels is grossly abnormal. A chimeric channel comprising LRRC8A and the first extracellular loop (EL1) of LRRC8C, 8A-8C(EL1), has similar abnormal DCPIB pharmacology. Purified 8A-8C(EL1) protein also forms narrow-pore homohexameric channels. An EL1 arginine residue unique to LRRC8A, R103, was proposed to participate in DCPIB inhibition. The R103F mutation greatly reduces DCPIB inhibition of LRRC8A homohexameric channels but has no effect on LRRC8A(R103F)+LRRC8C heteromers. The equivalent EL1 mutation, L105F, also reduces DCPIB inhibition of 8A-8C(EL1) homohexameric channels. In contrast, the L105F mutation has no effect on DCPIB inhibition of 8C-8A(IL125) homoheptameric channels. LRRC8 channels most likely evolved from pannexins, which have a well-established heptameric structure. Based on our functional and structural data and the evolutionary origin of LRRC8 proteins, we propose that native LRRC8/VRAC channels have a heteroheptameric configuration that gives rise to a large pore diameter that accounts for their high organic solute permeability.