Structure-function analysis of the volume-regulated anion channel VRAC using novel LRRC8 chimeras

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PROJECT SUMMARY The volume-regulated anion channel (VRAC) is expressed ubiquitously in vertebrate cells where it mediates the efflux of Cl- and organic solutes required for cell volume regulation, an essential physiological process. VRACs are activated and inactivated by cell swelling and shrinkage, respectively. They also detect changes in intracellular ionic strength, which modifies their sensitivity to cell volume changes. VRACs and the genes that encode them are implicated in multiple diseases including diabetes, obesity, cancer and immunity. Whole genome RNA interference screening led to the demonstration in 2014 that VRACs are encoded by five members of the Lrrc8 gene family, Lrrc8a–e. VRAC/LRRC8 channels are hexaheteromers and require co-assembly of the essential subunit LRRC8A with one or more other LRRC8 proteins. Subunit assembly order and stoichiometry are unknown. Cryo-electron microscopy (EM) structures of homomeric LRRC8A and LRRC8D channels were recently determined. However, LRRC8A and LRRC8D homomers do not exist in Nature. Furthermore, LRRC8A homomeric channels have non-native functional properties and LRRC8D channel properties are undefinable because they are not trafficked to the plasma membrane. Existing cryo-EM structures thus have limitations for understanding VRAC/LRRC8 structure-function relationships. Directly translating LRRC8A and LRRC8D cryo-EM structural information into functional understanding is further constrained by the unknown and likely variable stoichiometry and assembly of hexaheteromeric VRAC/LRRC8 channels. Our laboratory, funded by DK51610, has studied VRAC extensively and was the first to demonstrate many of the channel's unique functional properties. Most recently, we described novel LRRC8 chimeric channel constructs that allow detailed molecular study of homomeric channels with physiologically relevant functional properties and defined stoichiometry and assembly. Our chimera studies uniquely demonstrated that 1) the LRRC8A intracellular loop, IL1, has unique structural features, 2) it is required for cell volume sensing, 3) the LRRC8 C-terminus is required for sensing changes in intracellular ionic strength and 4) both the LRRC8A IL1 and C-terminus are required for correct cellular processing of VRAC/LRRC8 channels. The overarching goal of this R01 renewal application is to utilize these novel LRRC8 chimeras to better elucidate VRAC/LRRC8 channel structure-function relationships. We will characterize the roles of the LRRC8A C-terminus in VRAC/LRRC8 channel regulation and will test the hypothesis that the LRRC8 IL1 determines VRAC/LRRC8 channel pore properties and regulates channel gating. We will also determine the cryo-EM structure of a unique LRRC8 chimera in multiple physiologically relevant conformations. Our studies will provide novel insights into the regulation and function of VRAC/LRRC8 channels and will provide a higher confidence foundation for detailed mutagenesis-based structure-function analyses.