Identification of LRRC8 Heteromers as an Essential Component of the Volume-Regulated Anion Channel VRAC

Abstract

Abstract:Regulation of cell volume is critical for many cellular and organismal functions, yet the molecular identity of a key player, the volume-regulated anion channel VRAC, has remained unknown. A genome-wide siRNA screen in mammalian cells identified LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 multispan membrane proteins.Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A co-transfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Our work shows that VRAC defines a class of anion channels, suggests that VRAC is identical to the volumesensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents. One Sentence Summary:We show that the swelling-activated anion channel VRAC represents a structurally new class of anion channels that also conducts organic osmolytes. Main Text:Cells regulate their volume to counteract swelling or shrinkage caused by osmotic challenges and during processes like cell growth, division, and migration. As water transport across cellular membranes is driven by osmotic gradients, cell volume regulation requires appropriate changes of intracellular concentrations of ions or organic osmolytes like taurine (1, 2). Regulatory volume decrease (RVD) follows the extrusion of intracellular Cl -and K + and other osmolytes across the plasma membrane. A key player is the volume-regulated anion channel VRAC that mediates characteristic swelling-activated Cl --currents (I Cl(swell) ) and is ubiquitously expressed in vertebrate cells (3-5). Nearly inactive under resting conditions, VRAC slowly opens upon hypotonic swelling. The mechanism behind VRAC opening remains enigmatic. VRAC currents are outwardly rectifying (hence the alternative name VSOR for volume-stimulated outward rectifier (4, 5)) and show variable inactivation at insidepositive voltages. VRAC conducts iodide better than chloride and might also conduct organic osmolytes like taurine (6) (hence VSOAC, volume-stimulated organic osmolyte/anion channel (7)), but this notion is controversial (8-10). VRAC is believed to be important for cell volume regulation and swelling-induced exocytosis (11), and also for cell cycle regulation, proliferation and migration (1,3,4). It may play a role in apoptosis and various pathological (Fig. 1F). We hypothesized that VRAC contains LRRC8A as part of a heteromer and that LRRC8A overexpression led to a subunit stoichiometry that was incompatible with channel activity. LRRC8A has four closely related homologs (LRRC8B -LRRC8E) which all have four predicted transmembrane domains (19,20). EST databases suggested that all homologs were widely expressed.Immunocytochemistry of transfected HeLa cells ( fig. S4A) and of native HEK cells (Fig. 1, G and H) detected LRRC8A at the plasma membrane. Truncation of its carboxy-terminus as in a pat...

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

“…Notably, however we found no evidence for delayed astrocyte maturation or astrogliosis in both Mlc1 ‐null18 and Glialcam ‐null mice (not shown). LRRC8A has recently been identified as an essential component of the mammalian VRAC 35, 36. We found no differences in LRRC8A expression and subcellular localization between wild‐type, Glialcam ‐null and Mlc1 ‐null mice, which does not exclude a functional interaction between the related proteins.…”

“…We extended the immunohistochemical and immuno‐EM analysis to expression of the caveolar lipid raft protein caveolin‐1 and the recently identified VRAC channel component LRRC8A in wild‐type, Glialcam ‐null and Mlc1 ‐null mice 18, 35, 36. We found no differences between mutant and control animals (Fig.…”

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

“…[6][7][8][9][10][11][12] In 2014, 2 research groups independently reported that the leucine-rich repeat containing 8A (LRRC8A), which has 4 transmembrane domains and a leucine-rich repeat (LRR) domain at the C-terminus, is a core factor of VSOR in human cells. 13,14 They reported in common that knockdown of LRRC8A diminished endogenous VSOR currents in human cells, and such reduced currents could be rescued by introduction of exogenous LRRC8A, but overexpression of LRRC8A alone in normal cells paradoxically reduced endogenous VSOR currents. 13,14 Also, VSOR was shown to be a heteromeric channel containing not only LRRC8A but also other LRRC8 family members.…”

“…13,14 They reported in common that knockdown of LRRC8A diminished endogenous VSOR currents in human cells, and such reduced currents could be rescued by introduction of exogenous LRRC8A, but overexpression of LRRC8A alone in normal cells paradoxically reduced endogenous VSOR currents. 13,14 Also, VSOR was shown to be a heteromeric channel containing not only LRRC8A but also other LRRC8 family members. 13 Indeed, a multimeric complex mainly of these members with a molecular mass of »800 kDa was found to be sufficient to form the osmo-and ionic strength-sensitive anion channels in lipid bilayers.…”

“…13,14 Also, VSOR was shown to be a heteromeric channel containing not only LRRC8A but also other LRRC8 family members. 13 Indeed, a multimeric complex mainly of these members with a molecular mass of »800 kDa was found to be sufficient to form the osmo-and ionic strength-sensitive anion channels in lipid bilayers. 15 More recently, Jentsch's group has discovered the roles of LRRC8D in cisplatin and taurine transport and in cisplatin resistance acquirement in human KBM7 cells.…”