The Volume regulated anion channel (VRAC) is a key player in the cell-intrinsic regulatory processes which tend to restore the original cell volume upon osmotic challenges. This is achieved mediating the swelling-induced release of halide ions and organic osmolytes that subsequently drive water efflux through the membrane. VRAC is ubiquitously found in all vertebrate cells. In addition to its role in volume regulation, a large amount of literature suggests its importance in various other physiological and pathophysiological processes including cell proliferation, migration and apoptosis1. The molecular entity of VRAC was unknown until 2014, when two groups, using similar genome-wide siRNA screens, independently identified the leucine-rich repeat-containing protein 8A (LRRC8A) as an essential VRAC subunit 2, 3. Voss et al.2 further found that VRAC is a heteromer composed of closely related members of the LRRC8 family (LRRC8A-E), containing the obligatory LRRC8A subunit and at least one among the LRRC8B-E subunits. Recently, 4 Cryo-EM structures revealed the general architecture of VRAC by determining the structure of the homomeric LRRC8A complexes4-7. While homomeric LRRC8A channels are not physiologically relevant, the general architectures is likely conserved for heteromeric channels. The proteins are hexamers, in which four transmembrane domains of each subunit contribute to the formation of a relatively loosely packed, membrane imbedded, ion pore, followed by a cytoplasmic leucine rich repeat domain, that, as predicted from its sequence, consists of 16 structural repeats, each containing a β-strand followed by an α-helix. In three of the four published structures, the LRR domains assembled as trimers of dimer, while the fourth structure exhibited hexameric symmetry. One of the leading hypotheses to explain how the channel responds to a hypotonic stimulus is the sensing of a low intracellular ionic strength1. However, the mechanism of activation is not fully resolved and little is known about the actual, physiologically relevant stoichiometry. Indeed, it is known that the subunit composition of VRACs influences the biophysical properties of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics1. Also ROS sensitivity is strictly subunit specific; using fluorescently tagged LRRC8 proteins in oocytes it was found that LRRC8A-LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A-LRRC8C and LRRC8A-LRRC8D heteromers are inhibited1. The aim of this project is to provide deeper insight into the mechanism of activation of VRAC with particular emphasis on the role of the intracellular LRR domains. To address this major point, first we investigated the role of oxidation, transfecting different subunits in a cell line in which all 5 LRRC8 genes had been knocked out. Interestingly we observed that oxidation through the application of Chloramine T was sufficient to activate LRRC8A-LRRC8E heteromeric channels even in the absence of a pre-volume stimulation. To elucidate if oxidation acts directly on the LRRC8 channel-forming proteins or on regulatory factors we exploited two positively charged molecules known to reacts with cysteine residues and blocks their further oxidation. The inclusion of MTSET(+) or MTSES(-) in the pipette solution led to a dramatic increase of LRRC8A/LRRC8E mediated currents, followed by a complete inactivation of the channel. The subsequent application of hypotonic solution and Chloramine-T failed to reactivate the channels, suggesting that activation of LRRC8A/LRRC8E by chloramine-T is due to the oxidation of intracellular cysteines. Our results show that LRRC8 channels are directly modulated by oxidation in a subunit-dependent manner, confirming what was previously observed in oocytes. We are currently systematically mutating all intracellular cysteines of LRRC8E hoping to obtain insight on the specific residues responsible of the different effects mediated by ROS respectively on LRRC8C and LRRC8E.