Abstract

Volume-regulated anion channels (VRAC) are chloride channels activated in response to osmotic stress to regulate cellular volume and also participate in other cellular processes, including cell division and cell death. Recently, members of the LRRC8 family have been identified as the main contributors of VRAC conductance. LRRC8/VRAC is permeable to chloride ions but also exhibits significant permeability to various substrates that vary strongly in charge and size. In this study, we explored the intriguing ability of LRRC8/VRAC to transport glutathione (GSH), the major cellular reactive oxygen species (ROS) scavenger, and its involvement in epithelial-to-mesenchymal transition (EMT), a cellular process in which cellular oxidative status is a crucial step. First, in HEK293-WT cells, we showed that a hypotonic condition induced LRRC8/VRAC-dependent GSH conductance (PGSH/PCl of ~0.1) and a marked decrease in intracellular GSH content. GSH currents and GSH intracellular decrease were both inhibited by DCPIB, an inhibitor of LRRC8/VRAC, and were not observed in HEK293-LRRC8A KO cells. Then, we induced EMT by exposing renal proximal tubule epithelial cells to the pleiotropic growth factor TGFβ1, and we measured the contribution of LRRC8/VRAC in this process by measuring (i) EMT marker expression (assessed both at the gene and protein levels), (ii) cell morphology and (iii) the increase in migration ability. Interestingly, pharmacologic targeting of LRRC8/VRAC (DCPIB) or RNA interference-mediated inhibition (LRRC8A siRNA) attenuated the TGFβ1-induced EMT response by controlling GSH and ROS levels. Interestingly, TGFβ1 exposure triggered DCPIB-sensitive chloride conductance. These results suggest that LRRC8/VRAC, due to its native permeability to GSH and thus its ability to modulate ROS levels, plays a critical role in EMT and might contribute to other physiological and pathophysiological processes associated with oxidative stress.

Highlights

  • Volume-regulated anion channels (VRACs) are ubiquitously expressed proteins that are known to be activated during cell swelling[1]

  • LRRC8/VRAC activation is known to be triggered by hypotonic cell swelling

  • DCPIB alone had no significant effect on reactive oxygen species (ROS) production compared to the control. These results suggest that TGFβ1-induced ROS production might be due to an efflux of reduced GSH mediated by LRRC8/VRAC

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Summary

Introduction

Volume-regulated anion channels (VRACs) are ubiquitously expressed proteins that are known to be activated during cell swelling[1]. In the absence of osmotic challenge, VRACs are involved in different. LRRC8 subunits (LRRC8B-E) are necessary to form a constitutive channel[3]. VRACs are formed by hetero-hexamers of the different LRRC8 subunits in which LRRC8A is essential[3,4,5,6]. VRACs, similar to their homologous proteins pannexins, are poorly selective channels with a large pore that are permeable to diverse differently charged substrates varying strongly in size. The inhibitor DCPIB identified in 20029 was shown to be one of the most powerful molecules to target LRRC8/VRAC conductance[10]

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