Biology subjects

Nalwalk, J. W.

Publications and source records attributed to Nalwalk, J. W..

3 recordsLinked to original sources

bioRxiv2026

LRRC8A/D-containing anion channels mediate swelling-activated taurine release in the mouse brain

Volume-regulated anion channels (VRACs) are swelling-activated Cl- channels formed by heteromers of LRRC8 proteins (LRRC8A-E). VRACs mediate regulatory volume decrease by releasing Cl- and organic osmolytes, including taurine. In the brain, taurine functions as both an osmolyte and an inhibitory neuromodulator, acting as an agonist at glycine and GABAA receptors. Here, we used RNAi and genetically modified mice to (i) determine the contribution of LRRC8 subunits to swelling-activated taurine release, and (ii) test whether brain VRACs contribute to systemic osmoregulation. In astrocyte cultures lacking the essential VRAC subunit LRRC8A, swelling-activated [3H]taurine release was reduced by ~90%. Similarly, in vivo brain microdialysis revealed a [≥]90% reduction in swelling-activated taurine release in brain-wide LRRC8A knockout mice (bLRRC8A KO). RNAi experiments further confirmed LRRC8D as a key determinant of VRAC-mediated taurine release, with Lrrc8d knockdown reducing swelling activated [3H]taurine efflux by >50%. RNA-seq revealed enrichment of Lrrc8d in the hypothalamus, where astrocytic taurine release is proposed to inhibit magnocellular neurons and suppress arginine vasopressin secretion during hypoosmotic conditions. We therefore tested whether disrupting brain VRACs impairs recovery from systemic hypoosmotic stress. bLRRC8A KO mice and mice with gene trap-mediated LRRC8D depletion were challenged with an intraperitoneal injection of sterile water (7.5% body weight, under anesthesia), and serum osmolality was monitored over time. Unexpectedly, neither model showed impaired recovery from hypoosmotic challenge. Overall, this study identifies LRRC8A/D-containing VRACs as the primary conduit for swelling-activated taurine release in the brain, yet indicates that this pathway is not essential for maintaining systemic fluid-electrolyte homeostasis.

neuroscience↗

bioRxiv2025

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.

neuroscience↗

bioRxiv2022

Conditional deletion of LRRC8A in the brain protects against stroke damage independently of effect on swelling-activated glutamate release

The ubiquitous volume-regulated anion channels (VRACs), which are composed of LRRC8 proteins, facilitate cell volume homeostasis, and contribute to many other physiological processes. Prior studies demonstrated that treatment with non-specific VRAC blockers, or brain-specific deletion of the essential VRAC subunit LRRC8A, are highly protective in rodent stroke. In this work, we tested the widely accepted idea that harmful effects of VRACs in the brain are mediated by pathological release of the excitatory transmitter glutamate. We used two molecular genetic strategies to ablate LRRC8A expression in either brain astrocytes only (inducible deletion of Lrrc8aflox/flox with Aldh1l1CreERT2) or the majority of brain cells (neurons, astrocytes, and oligodendrocytes with NestinCre). To produce stroke, genetically modified mice were subjected to a 40-minute occlusion of the middle cerebral artery. The inducible deletion of astrocytic LRRC8A yielded no histological or behavioral protection. In contrast, the brain-wide LRRC8A knockout reduced ischemic infarction by ~50% in both heterozygotes (Het) and the full Lrrc8a knockout (KO) as compared to the control Lrrc8aflox/+ genotype. However, despite identical brain damage, Het and KO mice dramatically differed in their VRAC activities. Het mice had full swelling-activated glutamate release, while KO animals showed its virtual absence. These new findings refute the notion that VRAC-mediated glutamate release plays significant role in ischemic brain injury.

neuroscience↗