Ca(2+) mobilization-dependent reduction of the endoplasmic reticulum lumen is due to influx of cytosolic glutathione

BACKGROUND: The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca(2+) store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca(2+) from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear. RESULTS: We found that Ca(2+) mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathione-centered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca(2+) efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca(2+)-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca(2+) mobilization. While the action of puromycin was ascribable to Ca(2+) leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear. CONCLUSIONS: Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca(2+) mobilization. We postulate the existence of a Ca(2+)- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress.