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Open AccessArticle

Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting

1
Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany
2
Department of Human Medicine, MSH Medical School Hamburg, 20457 Hamburg, Germany
*
Author to whom correspondence should be addressed.
These authors contributed equally to the work.
Int. J. Mol. Sci. 2019, 20(23), 5879; https://doi.org/10.3390/ijms20235879
Received: 1 October 2019 / Revised: 11 November 2019 / Accepted: 21 November 2019 / Published: 23 November 2019
(This article belongs to the Special Issue Channels and Transporters in Cells and Tissues)
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. View Full-Text
Keywords: volume-regulated anion channel VRAC; volume-sensitive organic outward rectifying channel VSOR; leucine-rich repeat-containing; anion channel; subunit stoichiometry; heteromeric complex; tissue-specific protein expression; recombinant protein volume-regulated anion channel VRAC; volume-sensitive organic outward rectifying channel VSOR; leucine-rich repeat-containing; anion channel; subunit stoichiometry; heteromeric complex; tissue-specific protein expression; recombinant protein
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Pervaiz, S.; Kopp, A.; von Kleist, L.; Stauber, T. Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting. Int. J. Mol. Sci. 2019, 20, 5879.

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