Subunit-specific roles of LRRC8 proteins in determining glutamate permeability of astrocytic volume-regulated anion channels

Volume-regulated anion channels (VRACs) are ubiquitous chloride channels that play a crucial role in cell volume regulation but are also involved in many other physiological processes. VRACs are heteromers of proteins from the leucine-rich repeat-containing family 8 (LRRC8A-E), with LRRC8A being essential. Other LRRC8 subunits are expressed in a cell type-specific manner and modulate the biophysical properties of VRACs, including permeability to small signaling molecules. Here, we used primary astrocyte cultures from wild-type and genetically modified C57BL/6 mice to investigate (i) LRRC8 subunit composition of endogenous VRACs in the brain and (ii) the subunit determinants of VRAC permeability to the excitatory neurotransmitter glutamate. qPCR and RNA-seq revealed high expression of Lrrc8a-d in mouse forebrain and astrocytes. As expected, Lrrc8a deletion abolished VRAC activity, measured as swelling- activated release of the glutamate analogue D-[³H]aspartate. RNAi knockdown of individual subunits established that LRRC8A and LRRC8C are key components of astrocytic glutamate- permeable VRACs, with their siRNAs reducing radiotracer release by 85% and 56%, respectively. Downregulation of LRRC8D had a moderate effect, which depended on the severity of swelling. Combined silencing of LRRC8C and LRRC8D suggested that these subunits act in distinct channel populations. LRRC8B silencing alone was ineffective but partially rescued glutamate release in LRRC8C- or LRRC8D-knockdown cells. Overall, these findings indicate that astrocytic glutamate-permeable VRACs are primarily composed of LRRC8A and LRRC8C, with a possible structural role for LRRC8B. The refined understanding of VRAC subunit composition may inform the development of targeted VRAC inhibitors to mitigate glutamate excitotoxicity in neurological disorders. We examined the subunit composition of volume-regulated anion channels (VRACs), which contribute to pathological glutamate release in various neurological disorders. VRACs are heteromers formed by LRRC8A-E proteins, but the subunit configuration and functional roles within native channels remain poorly understood. In mouse astrocytes, we determined that glutamate-permeable VRACs are composed primarily of LRRC8A and LRRC8C, with structural support from LRRC8B. These findings provide a foundation for developing subunit-specific VRAC inhibitors as potential neuroprotective therapies.