Oligomerization States Of Lrrc8a And Lrrc8b, Essential Components Of The Volume-Regulated Anion Channel Vrac

Cellular volume regulation plays an important role in many cellular processes. VRAC is crucial for regulating volume decrease by allowing a net efflux of chloride and organic anions. Its molecular nature remained unclear until 2014 when Qiu et al. (Cell 157, 447-458) and Voss et al. (Science 344, 634-638) identified leucine-rich repeat-containing 8 (LRRC8) membrane proteins as subunits of VRAC. LRRC8 proteins, which occur in five isoforms (LRRC8A-E), have been suggested to share a common ancestor and accordingly a hexameric quaternary structure with pannexins (Abascal u0026 Zardoya, BioEssays 34, 551-560, 2012). Here, we expressed two human isoforms, hLRRC8A and hLRRC8B, in Xenopus laevis oocytes, purified the LRRC8 proteins by Ni-NTA chromatography in dodecyl maltoside, and assessed their oligomeric structure by blue native polyacrylamide gel electrophoresis (BN-PAGE). Detergent screening revealed that maltoside detergents with alkyl chain length from 10 to 12 were similarly well suited to purify hLRRC8A as a stable homomultimeric protein complex. The non-denatured homomeric hLRRC8A protein and the heteromeric hLRRC8A+B protein (with calculated non-glycosylated protomer masses of 95 and 92 kDa, respectively) approximately co-migrated in the BN-PAGE gel with the GFP-tagged tetrameric hTRPV1 channel (calculated mass close to 500 kDa). Slight denaturing treatments of the heteromeric hLRRC8A+B protein complex resulted in the appearance of lower order oligomers in the BN-PAGE gel, which we could tentatively identify as dimers, tetramers and hexamers by comparison with the migration patterns of concatenated hLRRC8A/A and A/B dimers and A/A/A trimers. We conclude that hLRRC8A and hLRR8A+B assemble as homo- and heterohexamers, respectively, in X. laevis oocytes. In contrast to the hLRRC8A isoform, hLRRC8B requires co-assembly with hLRRC8A to achieve a defined (hexameric) assembly state and plasma membrane localization.