A LRRC8 chimera with native functional properties is a heptamer with a large lipid-blocked pore

The volume-regulated anion channel VRAC mediates volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with an undefined, variable, and experimentally uncontrollable stoichiometry. The heteromeric structure of VRACs greatly complicates structure-function analyses. We circumvented this serious limitation by developing homomeric LRRC8 chimeric channels that recapitulate the functional properties of native heteromeric VRACs. Cryo-EM structures of abnormally functioning LRRC8A and non-functional LRRC8D homomeric channels demonstrated that they have a narrow-pore hexameric structure. In striking contrast, the 8C-8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A is a heptamer of asymmetrically arranged subunits. Five conformationally distinct structures from a single dataset exhibit diverse subunit arrangements and interactions, especially in the cytoplasmic LRR domains. The narrowest region of the 8C-8A(IL125) pore has a solvent-accessible radius of 4.7 Å. This limiting radius is similar to that estimated for native VRACs and is more than twice that of the limiting radii of homohexameric LRRC8A and LRRC8D channels. The 8C-8A(IL125) chimera is strongly inhibited by DCPIB, similar to that of native VRACs. In contrast, homohexameric LRRC8A channels exhibit grossly abnormal DCPIB pharmacology indicating a non-native pore structure. Within the 8C-8A(IL125) pore, we observe two layers of lipids running parallel to the membrane plane. Gaps between protomers are occupied by lipid molecules providing a potential route for lipids to move in and out of the channel pore. Like native VRACs, 8C-8A(IL125) chimeric channels are activated strongly by cell swelling and low intracellular ionic strength. We suggest that VRACs are heptamers similar to closely related pannexin channels and that movement of lipids into and out of the channel pore plays a central role in channel gating.