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.

In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that, besides the osmolytes taurine and myo-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and γ-aminobutyric acid (GABA) and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like myo-inositol, taurine and GABA, and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease the transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced the substrate preference of VRAC. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential coimmunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction.