Abstract
Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.
Highlights
In response to a reduction in extracellular osmolarity, water moves following the gradient of osmolarity through aquaporin water channels [1]
It was hypothesized that volume-regulated anion channels (VRACs) may be a heteromer of different proteins, and overexpression of one component in cell lines may lead to a stoichiometry that could be incompatible with VRAC activity [11]
Considering these small caveats, we believe that the expression of the fluorescently-tagged subunits, which are active in isotonic conditions, could be a useful tool to investigate the functional properties of the activity induced by LRRC8 proteins
Summary
In response to a reduction in extracellular osmolarity (or increase in intracellular osmolarity), water moves following the gradient of osmolarity through aquaporin water channels [1]. As happens many times in science, two groups identified and published their discoveries at the same time using the same strategy [10,11]: a genome-wide RNA interference screen using a fluorescence readout of VRAC activity (using previously developed anion-sensitive yellow fluorescent proteins [12]). Another group performed a similar type of screening in Drosophila cells and identified Bestrophin [13], but no LRRC8 proteins, since they are not present in Drosophila. Consistent with LRRC8A being a VRAC component, expressed LRRC8A was localized at the plasma membrane after transient transfection in cell lines [10,11]
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