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...

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Canonical volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have many other important roles, including tumor drug resistance and release of neurotransmitters. Although VRAC-mediated swelling-activated chloride currents (I Cl,vol ) have been studied for decades, exploration of the structure-function relationship of VRAC has become possible only after the recent discovery that VRACs are formed by differently composed heteromers of LRRC8 proteins. Inactivation of I Cl,vol at positive potentials, a typical hallmark of VRACs, strongly varies between native cell types. Exploiting the large differences in inactivation between different LRRC8 heteromers, we now used chimeras assembled from isoforms LRRC8C and LRRC8E to uncover a highly conserved extracellular region preceding the second LRRC8 transmembrane domain as a major determinant of I Cl,vol inactivation. Point mutations identified two amino acids (Lys-98 and Asp-100 in LRRC8A and equivalent residues in LRRC8C and -E), which upon charge reversal strongly altered the kinetics and voltage dependence of inactivation. Importantly, charge reversal at the first position also reduced the iodide > chloride permeability of I Cl,vol . This change in selectivity was stronger when both the obligatory LRRC8A subunit and the other co-expressed isoform (LRR8C or -E) carried such mutations. Hence, the C-terminal part of the first extracellular loop not only determines VRAC inactivation but might also participate in forming its outer pore. Inactivation of VRACs may involve a closure of the extracellular mouth of the permeation pathway.Volume-regulated anion channels (VRACs) 4 are an important part of the molecular machinery involved in cellular volume homeostasis. They open upon cell swelling and allow for the extrusion of intracellular chloride and osmolytes during regulatory volume decrease (1-3). The current ascribed to VRACs, termed volume-activated anion current (I Cl,vol ), is found in all vertebrate cell types (4). Besides their well described role in cell volume regulation, VRACs have been implicated in many physiological processes, such as cell migration and proliferation, apoptosis, cell cycle maintenance, and release of extracellular signaling molecules (1, 4 -6). Further evidence linked VRACs to several pathologies, such as cancer and ischemic stroke (5, 7). However, the lack of knowledge of the underlying protein(s) forming these channels has greatly hampered further exploration and verification of their suggested physiological and pathophysiological roles. Only recently, LRRC8 (leucine-rich repeat-containing 8) family proteins, which display four putative transmembrane domains and a large C terminus with up to 17 leucine-rich repeats, have been identified as essential components of VRACs (8, 9), and LRRC8 heteromers very likely form the channel pore (6, 8, 10). The physiological importance of VRACs is underscored by the drastically increased lethality of constitutive Lrrc8a KO mice that display multiple tissue abnormalities (11). Mo...