Abstract
The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents.
Highlights
Cells exploit various channels and transporters for inorganic ions and organic osmolytes to osmotically adjust their volume according to their physiological needs [1,2]
Membrane depolarization by volume-regulated anion channel (VRAC)-mediated Cl- efflux upon glucose-induced cell swelling contributes to insulin secretion in pancreatic β-cells [11,12,13] and VRAC is involved in membrane potential alterations during myogenic differentiation [14]
We found that LRRC8A is present at low levels, suggesting that VRAC complexes may contain only one or very few of this essential subunit
Summary
Cells exploit various channels and transporters for inorganic ions and organic osmolytes to osmotically adjust their volume according to their physiological needs [1,2]. The substrate specificity of VRAC alters with the subunit composition, as revealed by heterologous expression of LRRC8 combinations and/or using gene-depleted cells lines. While there is information about the relative protein levels of a given LRRC8 paralogue between tissues, relative expression levels of the different subunits within a cell type are only available on mRNA level [35,36,41], despite the potentially high physiological relevance of the subunit ratio. We describe a method using quantitative immunoblotting to measure the absolute protein amount and, importantly, the relative protein levels of the different VRAC LRRC8 subunits in a given cell type or organ. Epitope Peptide QRTKSRIEQGIVDRSE [22] QSLPYPQPGLESPGIESPT [7] EDALFETLPSDVREQMKAD [7] LEVKEALNQDVNVPFANGI [7] LYEGLPAEVREKMEEE [22]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call