Reversible glutathionylation of the plasmalemmal Ca2+‐ATPase/pump by the thiol‐oxidizing agent diamide

Abstract

In many cells, oxidative stress (OS) causes a rise in cytosolic free Ca2+ concentration ([Ca2+]i) often leading to necrotic cell death. During OS, proteins can be modified at specific cysteine residues by glutathione (GSH). To determine if the plasmalemmal Ca2+‐ATPase/pump (PMCA) is glutathionylated during OS, membranes from PMCA4‐expressing Sf9 cells or purified PMCA were treated with diamide and biotinylated GSH (biotin‐GSH). Glutathionylated proteins were captured by streptavidin beads and probed for PMCA. Treatment with biotin‐GSH (125 μM) alone resulted in little or no glutathionylation of PMCA. However, robust glutathionylation was observed within 10 minutes in the presence of diamide (100 μM). The reaction was reversed by DTT or glutaredoxin. Native PMCAs in membranes from mouse heart and from cultured bovine aortic endothelial cells (BAEC) were also glutathionylated in vitro. Additionally, PMCA was glutathionylated in response to diamide in intact BAECs loaded with BioGEE, a membrane permeable form of biotin‐GSH. Lastly, glutathionylation of PMCA4 reduced Ca2+‐ATPase activity 29.9 ± 8.9% (n=8). These results demonstrate that PMCA is a target for glutathionylation, and that the modified PMCA has a reduced activity. Inhibition of PMCA by glutathionylation could explain, at least in part, the rise in basal [Ca2+]i that occurs in endothelial cells during periods of OS. Supported in part by HL097355.