VRAC: unravelling the complexity of LRRC8 subunit regulation by oxidation.

Abstract

To maintain ion homeostasis, cells must regulate their volume. Water flux is controlled through the exchange of ions and osmolytes between the cytosol and the extracellular compartment. Under hypotonic conditions, when the cytosol osmolality is higher than the extracellular milieu, water flows into the cells leading to rapid cell swelling. This water influx decreases intracellular osmolality and increases cell volume, thereby compromising cellular functions. To prevent damage resulting from cell swelling, a process called regulatory volume decrease (RVD) is activated. This consists of an efflux of ions and osmolytes that secondarily drives water efflux and a return to a physiological cellular volume. One of the main actors of RVD is an anion channel known as the volume-regulated anion channel (VRAC). The VRAC current was first recorded in the 1980s in lymphocytes but was rapidly shown to be present in virtually all animal cells. It is characterized by permeability to chloride but also to other anions and osmolytes such as bicarbonate, taurine and glutamate. VRAC is also known to be activated in a process called apoptotic volume decrease (AVD), which leads to a volume decrease in the absence of cell swelling or change in osmolality. The search for the molecular identity of VRAC has lasted a very long time with many candidates. The difficulty arose from the complexity of VRAC's biophysical properties, which the proposed candidates failed to completely fulfil. Furthermore, the different mechanisms triggering VRAC activation during RVD or AVD remain unknown and are still under investigation. Finally, in 2014 two independent teams claimed that the protein leucine-rich repeat containing 8A (LRRC8A) is essential for VRAC current (Qiu et al. 2014; Voss et al. 2014). In fact, VRACs are hetero-hexamers of LRRC8A and at least one other LRRC8 isoforms (LRRC8B–LRRC8E) (Voss et al. 2014). Far from ending the VRAC mystery, researchers are now trying to understand the mode of assembly of the different LRRC8 subunits, their complex regulations and their physiological roles. Even if there is no clue as to the stoichiometry of LRRC8 subunit assembly, some features are starting to become clear. Heteromers of LRRC8A–C or LRRC8A–E present a high chloride channel activity that is less marked for the LRRC8A–D combination (Lutter et al. 2017). LRRC8A–E and to a lesser extent LRRC8A–C support fluxes of negatively charged organic ions such as aspartate. In contrast, LRRC8A–D is implicated in the transport of a wide variety of compounds: negatively charged entities (aspartate), neutral molecules (taurine, serine and GABA) and even positively charged entities (lysine) and more intriguingly, large charged molecules such as cisplatin or blasticidin S (Jentsch et al. 2016). Strikingly, the LRRC8A–B heteromers exhibit no identified transport activity. Based on this substrate heterogeneity, it is tempting to believe that each subunit combination may have a particular function within the cell with specific regulation mechanisms. In this issue of The Journal of Physiology, Gradogna et al. (2017) shed light on the modulation of VRAC current by oxidation. They show opposite effects depending on the different combinations of LRRC8 subunits. Using heterologue expression in Xenopus oocytes, the authors have previously described that addition of a C-term tag on LRRC8 subunits generates a constitutively active VRAC conductance (Gaitán-Peñas et al. 2016). They now show, using these active constructions, that swelling-activated chloride currents from tagged LRRC8A–E are potentiated by oxidizing agents (chloramine-T and tert-butyl hydroperoxide). They also demonstrate that this regulation can occur in wild-type LRRC8A–E combinations and is dependent on the oxidation state of LRRC8 internal cysteines. In contrast, chloride currents generated by heteromers of LRRC8A–C or LRRC8A–D are inhibited by the oxidizing agent chloramine-T and slightly potentiated by the reducing agent DTT. They complemented their work by a study on lymphocytes which are characterized by low expression of LRRC8E and exhibit, therefore, an expected swelling-activated chloride current inhibited by oxidation. In conclusion, antagonistic effects of oxidation depend on LRRC8 subunit combinations (Fig. 1), which could explain some discrepancies in the literature. This breakthrough in the VRAC modulation by oxidation or reduction will certainly allow the scientific community to understand better the role of oxidation in the processes of both RVD and AVD. More importantly, it appears from the latest publications that scientists have never been as close to unravelling the complexity of the volume-regulated anion channel. Finally, there is not one VRAC but several LRRC8 heteromers possessing their own regulation mechanisms and functions. None declared. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.