Heteromeric LRRC8 channel assemblies and their impact on channel activity

Volume regulated anion channels (VRAC) are activated in response to the osmotic swelling of vertebrate cells to catalyze the efflux of anions and small osmolytes. VRACs are multimeric channels that are composed of leucine-rich repeat-containing 8 (LRRC8) proteins, a protein family encompassing five paralogues in mammals (LRRC8A-E). Endogenous VRACs are known to form heteromeric assemblies of LRRC8A with at least one further LRRC8 isoform. Until recently, only structures of homomeric LRRC8 channels were described, while the structures of heteromeric channels remained elusive. Combining results from targeted quantification mass spectrometry and cryoEM with proteins obtained from heterologous overexpression, we investigated the subunit stoichiometry and structure of heteromeric LRRC8A/C channels. For these proteins, we observed a ratio of LRRC8A:C subunits of 2:1, which was confirmed in cryo-EM structures where LRRC8A and LRRC8C subunits cluster as pairs in a hexameric protein assembly. This subunit arrangement increases the pore size in LRRC8A/C channels compared to homomeric LRRC8A. Moreover, we found a tight interaction between LRR domains of LRRC8A subunit pairs of heteromers, reflecting a similar arrangement observed in homomeric LRRC8A. Conversely, the same parts of LRRC8C subunits were not resolved, indicating increased flexibility of their cytosolic regions. The structure thus suggests that the LRRC8C subunits perturb the tight packing of the LRRC8A subunits in heteromeric channels. Similar destabilizing effects were previously observed in LRRC8A homomers binding synthetic nanobodies with activating phenotype. There the binders induced a destabilization of the LRR domain leading to an increase of the open probability of homomeric channels. Collectively, our results suggest that the incorporation of LRRC8C in heteromeric LRRC8A/C channels destabilizes the tight packing of LRRC8A subunits to improve the activation properties of the channels.