Molecular Mechanisms Underlying Oxidation Sensitivity of VRAC

Abstract

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. Recently identified as a hetero-hexamer formed by LRRC8 proteins, its mechanism of activation remains elusive. Among the relevant triggers identified till now, activation of VRAC has been linked to the action of reactive oxygen species. Using GFP tagged LRRC8 channels expressed in oocytes it was found that oxidation sensitivity is subunit specific: LRRC8A-LRRC8E (8A-8E) heteromers are activated by oxidation, whereas 8A-8C and 8A-8D heteromers are inhibited (Gradogna et al, J Physiol, 2017). Here, using LRRC8 knockout HEK cells, we confirmed oxidation mediated activation of 8A-8E and inhibition of 8A-8C heteromers in a system devoid of background of endogenous LRRC8 genes. To further elucidate the mechanism underlying oxidative activation of 8A-8E, we added charged cysteine reactive compounds (MTSET⁺ or MTSES⁻) to the pipette solution. Both led to an initial dramatic increase of 8A-8E currents, followed by a complete inactivation. Subsequent application of hypotonic solution and oxidizing chloramine-T failed to reactivate the channels, suggesting that activation of 8A-8E by chloramine-T is mediated by intracellular cysteines. This was confirmed by chimeras in which the leucine-rich repeats (LRRs) were swapped between 8E and 8C, resulting in a corresponding interchange of the respective oxidation sensitivity, demonstrating that the relevant residues are located in the LRRs. Taken together, these findings corroborate the hypothesis of a strictly subunit dependent mechanism. Successive subchimeras based on the logic of the bisection method will be used to precisely identify cysteine(s) responsible of oxidation-dependent activation and to deepen the understanding of the in vivo mechanism.