Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells

Calcium (Ca²⁺) is an essential mineral of endoplasmic reticulum (ER) luminal biochemistry because of the Ca²⁺ dependence of ER-resident chaperones charged with folding de novo proteins that transit this cellular compartment. ER Ca²⁺ depletion reduces the ability of chaperones to properly fold the proteins entering the ER, thus leading to an accumulation of misfolded proteins and the onset of a state known as ER stress. However, not all conditions that cause ER stress do so in a manner dependent on ER Ca²⁺ depletion. Agents such as tunicamycin inhibit the glycosylation of de novo polypeptides, a key step in the maturation process of newly synthesized proteins. Despite this established effect of tunicamycin, our understanding of how such conditions modulate ER Ca²⁺ levels is still limited. In the present study, we report that a variety of ER stress-inducing agents that have not been known to directly alter ER Ca²⁺ homeostasis can also cause a marked reduction in ER Ca²⁺ levels. Consistent with these observations, protecting against ER stress using small chemical chaperones, such as 4-phenylbutyrate and tauroursodeoxycholic acid, also attenuated ER Ca²⁺ depletion caused by these agents. We also describe a novel high-throughput and low-cost assay for the rapid quantification of ER stress using ER Ca²⁺ levels as a surrogate marker. This report builds on our understanding of ER Ca²⁺ levels in the context of ER stress and also provides the scientific community with a new, reliable tool to study this important cellular process in vitro.