Cell surface detection of LRRC8A subunit of the anion channels VRAC in native living human lymphoma cells U937 by flow cytometry

Abstract

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Volume regulated anion channels (VRAC) play significant role in cell biology. Much progress has been made in recent years in studying their molecular structure and electrophysiological properties. The regulation of VRAC at the cellular level in long-term scale in biological processes such as proliferation, differentiation and apoptosis remains poorly explored. Estimation of the abundance of the channels at the cell membrane in living cells is a necessary step in this work. VRAC subunit proteins were detected until now mostly in cells transfected with the somehow tagged VRAC subunits, by immunoblotting of the solubilized entire cells or cell membranes or by immunoimaging of the fixed cells. We explored whether this problem can be solved in native living cells using flow cytometry and commercially available antibody against an extracellular loop of the lRrC8A subunit known as obligatory for operating VRAC heteromeric channel. Human lymphoid cells U937 were used as an object and two types of cell alteration were tested which are supposed to be followed by an increase in chloride permeability of the cell membrane: an induction of apoptosis with staurospo-rine (STS) and cell water balance disturbance in hypotonic medium. It is found that endogenous LRRC8A subunits are detected and their abundance at the cell membrane of the native living U937 cells can be evaluated using flow cytometry and applied Alomone antibody. It appeared, however, that cell treatment for 1 h with either STS, or with hypotony does not cause visible change in the number of lRrC8A subunits expressed at the cell surface. We conclude that an increase in the entire chloride permeability of the cell membrane under considered conditions is not due to an increase in number of channels exposed at the cell membrane but alteration of channel properties.