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 important roles in cell volume regulation and numerous other physiological processes. VRACs are heteromeric complexes composed of leucine-rich repeat-containing proteins LRRC8A-E. LRRC8 subunit composition determines 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) subunit composition of native VRACs in the brain and (ii) subunit determinants of VRAC permeability to the excitatory neurotransmitter glutamate. qRT-PCR and RNA-seq analyses revealed high expression of Lrrc8a-d in mouse forebrain and astrocytes. Genetic deletion of the essential LRRC8A protein abolished swelling-activated glutamate release, measured as efflux of the non-metabolizable D-[3H]aspartate, confirming the crucial role of VRACs in this process. RNAi-mediated knockdown of individual subunits identified LRRC8A and LRRC8C as key components of glutamate-permeable astrocytic VRACs. qRT-PCR and Western blot analyses further showed that knockdown of LRRC8A or LRRC8C reciprocally altered the protein stability of the partner subunit without affecting their mRNA levels. A similar pattern of mutual regulation was observed between LRRC8A and LRRC8D. In contrast to LRRC8C, downregulation of LRRC8D had a more limited impact on glutamate release. Additional double-knockdown experiments demonstrated that LRRC8C- and LRRC8D-containing channels form distinct VRAC populations. This model was further supported by Western blot results showing no reciprocal regulation of LRRC8C and LRRC8D stability. Together, these findings refine our understanding of how the subunit organization of native brain VRACs governs gliotransmitter release, with implications for normal brain function and neurological disease. NEW & NOTEWORTHYVolume-regulated anion channels (VRACs) are ubiquitously expressed chloride channels composed of LRRC8A-E proteins. In human disorders, gain or loss of VRAC function leads to severe neurological phenotypes, potentially due to altered release of amino acid neurotrans-mitters. Here, we show that glutamate-permeable VRACs in brain astrocytes are primarily composed of LRRC8A and LRRC8C proteins. These findings provide insight into subunit organization of native VRACs in the CNS, with implications for normal brain function and neurological disease.