The volume-regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl /anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled d-[ C]aspartate and [ H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [ H]taurine and myo-[ H]inositol, without major impact on the simultaneously measured efflux of the charged d-[ C]aspartate. In contrast, the release of d-[ C]aspartate was preferentially sensitive to deletion of LRRC8C+LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D-containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules.
Regulation of cellular volume is a critical homeostatic process that is intimately linked to ionic and osmotic balance in the brain tissue. Because the brain is encased in the rigid skull and has a very complex cellular architecture, even minute changes in the volume of extracellular and intracellular compartments have a very strong impact on tissue excitability and function. The failure of cell volume control is a major feature of several neuropathologies, such as hyponatremia, stroke, epilepsy, hyperammonemia, and others. There is strong evidence that such dysregulation, especially uncontrolled cell swelling, plays a major role in adverse pathological outcomes. To protect themselves, brain cells utilize a variety of mechanisms to maintain their optimal volume, primarily by releasing or taking in ions and small organic molecules through diverse volume-sensitive ion channels and transporters. In principle, the mechanisms of cell volume regulation are not unique to the brain and share many commonalities with other tissues. However, because ions and some organic osmolytes (e.g., major amino acid neurotransmitters) have a strong impact on neuronal excitability, cell volume regulation in the brain is a surprisingly treacherous process, which may cause more harm than good. This topical review covers the established and emerging information in this rapidly developing area of physiology.
It is well known that the electrical signaling in neuronal networks is modulated by chloride (Cl) fluxes via the inhibitory GABA and glycine receptors. Here, we discuss the putative contribution of Cl fluxes and intracellular Cl to other forms of information transfer in the CNS, namely the bidirectional communication between neurons and astrocytes. The manuscript (i) summarizes the generic functions of Cl in cellular physiology, (ii) recaps molecular identities and properties of Cl transporters and channels in neurons and astrocytes, and (iii) analyzes emerging studies implicating Cl in the modulation of neuroglial communication. The existing literature suggests that neurons can alter astrocytic Cl levels in a number of ways; via (a) the release of neurotransmitters and activation of glial transporters that have intrinsic Cl conductance, (b) the metabotropic receptor-driven changes in activity of the electroneutral cation-Cl cotransporter NKCC1, and (c) the transient, activity-dependent changes in glial cell volume which open the volume-regulated Cl/anion channel VRAC. Reciprocally, astrocytes are thought to alter neuronal [Cl] through either (a) VRAC-mediated release of the inhibitory gliotransmitters, GABA and taurine, which open neuronal GABA and glycine receptor/Cl channels, or (b) the gliotransmitter-driven stimulation of NKCC1. The most important recent developments in this area are the identification of the molecular composition and functional heterogeneity of brain VRAC channels, and the discovery of a new cytosolic [Cl] sensor - the Wnk family protein kinases. With new work in the field, our understanding of the role of Cl in information processing within the CNS is expected to be significantly updated.
The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brainwide conditional LRRC8A knockout mice (LRRC8A bKO) using Nestin Cre -driven Lrrc8a flox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase.Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC
Volume‐regulated anion channel (VRAC) is a glutamate‐permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes – the cell type which shows prominent swelling in stroke – to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post‐ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin–luciferase ATP assays and a Seahorse analyzer. Swelling‐activated glutamate release was quantified with the radiotracer D‐[3H]aspartate. The specific contribution of VRAC to swelling‐activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine‐rich repeat‐containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT‐PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2‐deoxy‐D‐glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP‐depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty‐four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.