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...

Ca-activated Cl currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca-activated Cl channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl efflux. Here, we confirmed the co-localization of the Ca-activated Cl channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca-activated Cl currents. Although these currents persisted in the VNOs of mice lacking , they were undetectable in olfactory neuron-specific knockout mice irrespective of the presence of The loss of Ca-activated Cl currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male-male territorial aggression in olfactory / double knockout mice. We conclude that Ano1 mediates the bulk of Ca-activated Cl currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice.