Volume-regulated anion channel--a frenemy within the brain

Abstract

The volume-regulated anion channel (VRAC) is a ubiquitously expressed yet highly enigmatic member of the superfamily of chloride/anion channels. It is activated by cellular swelling and mediates regulatory cell volume decrease in a majority of vertebrate cells, including those in the central nervous system (CNS). In the brain, besides its crucial role in cellular volume regulation, VRAC is thought to play a part in cell proliferation, apoptosis, migration, and release of physiologically active molecules. Although these roles are not exclusive to the CNS, the relative significance of VRAC in the brain is amplified by several unique aspects of its physiology. One important example is the contribution of VRAC to the release of the excitatory amino acid neurotransmitters glutamate and aspartate. This latter process is thought to have impact on both normal brain functioning (such as astrocyte-neuron signaling) and neuropathology (via promoting the excitotoxic death of neuronal cells in stroke and traumatic brain injury). In spite of much work in the field, the molecular nature of VRAC remained unknown until less than 2 years ago. Two pioneer publications identified VRAC as the heterohexamer formed by the leucine-rich repeat-containing 8 (LRRC8) proteins. These findings galvanized the field and are likely to result in dramatic revisions to our understanding of the place and role of VRAC in the brain, as well as other organs and tissues. The present review briefly recapitulates critical findings in the CNS and focuses on anticipated impact on the LRRC8 discovery on further progress in neuroscience research.

Keywords: Astrocytes; Cell volume regulation; Central nervous system; LRRC8; Neurons; VRAC.

Fig. 1. Membrane mechanisms responsible for regulatory volume decrease (RVD) in osmotically swollen cells

LEFT: Under steady-state conditions, membrane permeability is dominated by K⁺ channels (outlined in box) and transmembrane water fluxes are equilibrated. RIGHT: In the response to osmotic swelling cells dramatically increase membrane permeability to Cl⁻ and other anions due to activation of volume regulated anion channels (VRAC, outlined in box). This facilitates cooperative loss of K⁺ and Cl⁻, which is coupled with release of osmotically obligated water. Many cells types additionally activate KCl loss via electroneutral K⁺, Cl⁻ transporters (KCC, also outlined in box). Another electroneutral transporter, NKCC1, drives the net uptake of Na⁺/K⁺/Cl⁻ under basal conditions, and is additionally strongly activated by cellular shrinkage. However, in at least one brain cell type, astrocytes, this transporter is paradoxically stimulated by cell swelling and contributes to sustained astrocytic edema in CNS pathologies (asterisk).

Fig. 2. Representative electrophysiology recordings of VRAC currents and swelling activated glutamate release in brain cells in vitro, and in brain cortex in vivo

A, Whole-cell recordings of Cl⁻ currents primary rat astrocytes exposed to hypoosmotic medium (−60 mOsm) with symmetrical concentrations of Cl⁻ in bath and pipette solutions. In the main panel, the time course Cl⁻ current development was recorded in response to brief alterative voltage steps to +/−40 mV from the holding potential of 0 mV. In the inset, Cl⁻ currents in response to voltage steps from −100 to +100 mV in 20 mV increments. Note outward rectification and time-dependent current inactivation at high positive potentials. Reproduced with permission from I.F. Abdullaev et al. (2006). B, Whole-cell recordings of VRAC Cl⁻ currents performed in rat primary microglial cells. The conditions and composition of experimental solutions were similar to those used in (A). Reproduced with permission from T.J. Harrigan et al. (2008). C, Swelling activated glutamate release from primary rat astrocytes traced with its non-metabolizable analogue d-[³H]aspartate. Astrocytes were superfused with isoosmotic or hypoosmotic (−100 mOsm) media in the presence or absence of the VRAC blocker DCPIB as indicated. ***p<0.001, effect of DCPIB on glutamate release in swollen cells. Modified with permission from I.F. Abdullaev et al. (2006). D, Swelling activated glutamate release from primary rat microglial cells measured with the non-metabolizable glutamate analogue d-[³H]aspartate. The experimental conditions were identical to those shown in (C). ***p<0.001, effect of DCPIB on glutamate release in swollen cells. Modified with permission from T.J. Harrigan et al. (2008). E, Hypoosmotic stimulation of glutamate release from the rat cortical tissue measured using a microdialysis approach. Cortex was perfused with isoosmotic or hypoosmotic artificial cerebrospinal fluid additionally containing the low affinity VRAC blocker DNDS, which is well tolerated in vivo. Collected microdialysis samples were analyzed off-line for the presence of L-glutamate and other amino acids using HPLC. *p<0.05, effect of DNDS on glutamate release under hypoosmotic conditions. Reproduced from R.E. Haskew-Layton et al. (2008) under the Creative Commons Attribution (CC BY) license.

Fig. 3. The VRAC inhibitor tamoxifen potently protects rat brain against damage in experimental stroke model, and reduces the intraischemic glutamate release in rat cortical tissue

A, Representative images of brain sections from rats subjected to 2-h experimental stroke and evaluated 3 days after ischemia. Animals were treated with vehicle (A), or the VRAC blocker tamoxifen (5 mg/kg) given i.v. either 10 min prior (B), or 3 h after initiation of stroke (C). At the end of experiment rats were euthanized and their brains were sliced and stained with triphenyltetrazolium chloride to visualize tissue damage (infarcted tissue is unstained). Reproduced with permission from H.K. Kimelberg et al. (2000). B, Dynamics of the microdialysate glutamate levels during and after focal cerebral ischemia. Vehicle control (5% DMSO), the VRAC blocker tamoxifen (50 µM), or the inhibitor of the glial glutamate transporter GLT-1 dihydrokainate (DHK, 1 mM) were delivered through the microdialysis probe placed in the ischemic penumbra. *p<0.05, tamoxifen vs. DMSO and DHK during ischemia; #p<0.05, DHK vs tamoxifen and control after ischemia (repeated measures ANOVA). Modified with permission from P.J. Feustel et al. (2004). C, Hypothetical representation of the processes in the ischemic penumbra, based on the results of microdialysis experiments. Pathological swelling of astrocytes (and perhaps other cells) triggers glutamate release via the VRAC. Increased glutamate levels cause Ca2⁺-dependent damage and death of neuronal cells due to excessive activation of neuronal NMDA receptors. In penumbra, glial transporter GLT-1 continues to take up extracellular glutamate and, therefore, addition of DHK leads to high sustained glutamate levels during and after ischemia.

Fig. 4. Schematic representation of the predicted topology of LRRC8A and multimeric composition of the LRRC8 heteromeric complexes forming VRAC

A, Transmembrane topology of the LRRC8A protein based on the model proposed by Abascal and Zardoya (2012). This diagram contains positions of known glycosylation sites (Y), four conserved cysteines (yellow circles), N-terminal leucine-rich repeat motifs (red cylinders), and putative phosphorylation sites for casein kinase II (CK), protein kinase C (PKC), and cAMP-/cGMP-dependent kinases (PKA/PKG) (color coded multipoint stars, as indicated). The putative phosphorylation sites were determined using LRRC8A sequence from NCBI Protein database (sequence NP_001120717.1) and ScanProsite phosphorylation probability software (Swiss Institute for Bioinformatics. B, Multimeric composition of LRRC8 complexes based on pannexin homology and findings of Voss et al. (2014). Six subunits are proposed to form a channel with one central pore, LRRC8A (red) is an obligatory component of this complex, and has to be heteromerized with at least one of the four other members of the same family (LRRC8B-E) to form a functional VRAC channel. The exact amount of each subunit in heteromer is unknown.

Fig. 5. LRRC8 proteins are expressed in primary rat astrocytes and form swellingactivated glutamate release pathway

A, Results of quantitative RT-PCR experiments quantifying mRNA expression levels for LRRC8A-E. Data are mean values of LRRC8 expression levels in 5 astrocyte cultures, as normalized to the housekeeping genes GAPDH, RPL13a, and RPS20. B, Swelling activated glutamate release from primary rat astrocytes measured with the non-metabolizable glutamate analogue d-[³H]aspartate. Astrocytes were transfected with either scrambled siRNA (siNC) or mix of 4 siRNAs targeting the pore forming LRRC8A (siLrrc8a_mix). Functional assays of glutamate release were performed 72 h after transfection. ***p<0.001, swelling-activated release in control cells and cells treated with LRRC8A siRNA. ^# p<0.05, release under basal conditions. Modified with permission from M.C. Hyzinski-Garcia et al. (2014).