Abstract
ABSTRACT Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)-induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.
Highlights
Chloride channels are key players in regulating ionic and water balance in animal cells, as chloride is the main external anion for most cells and chloride channels are the main electroconductive pathway for this ion through the cell membrane [1–3]
Because Volume regulated anion channel (VRAC) regulates chloride flux and cell volume responses, we investigated whether STS-induced apoptosis or hypotonic stress in U937 cells alter the expression of cell-surface VRAC using our validated flow cytometry method
Our results show that endogenous VRAC can be quantified in living cells with average native expression levels by flow fluorometry using the Alomone Lab antibody for the LRRC8A subunit
Summary
Chloride channels are key players in regulating ionic and water balance in animal cells, as chloride is the main external anion for most cells and chloride channels are the main electroconductive pathway for this ion through the cell membrane [1–3]. Volume regulated anion channel (VRAC) is a ubiquitously expressed chloride channel that has attracted much attention since the molecular structure of VRAC has been identified [4–6]. While the molecular structure of VRAC is well documented [18], understanding how VRAC expression at the membrane is regulated has been poorly explored due to lack of appropriate methodology. Methods for quantifying VRAC channels at the cell membrane are critical. The use of this antibody to quantify cell membrane VRAC expression on living cells has not been explored until now
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