Rapid quantification of intracellular calcium stores reveals effects of membrane micropeptides on SERCA function

To determine how regulation of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) affects the Ca²⁺ content of the endoplasmic reticulum (ER), we developed a ratiometric ER-localized Ca²⁺ indicator to rapidly quantify Ca²⁺ stores and assess SERCA function in live cells. This assay enables screening of membrane micropeptides and small molecules that modulate SERCA and Na⁺/K⁺-ATPase activity and may facilitate development of therapies that target cellular Ca²⁺ handling. Of the micropeptides tested, phospholamban (PLB) had the greatest degree of inhibition of SERCA, as measured by a decrease in ER Ca²⁺ content compared to control. Sarcolipin (SLN), endoregulin (ELN), and another-regulin (ALN) also decreased ER Ca²⁺ content, though less potently than PLB. We also investigated micropeptides that have been shown to have a positive effect on ER Ca²⁺ uptake. Dwarf open reading frame (DWORF), a positive modulator of SERCA activity, and phospholemman (PLM), an inhibitor of the Na⁺/K⁺-ATPase, both increased ER Ca²⁺ content compared to control. A superinhibitory variant of PLM, R70C, further increased ER Ca²⁺ load compared to wild type PLM. Overall, our findings indicate that the inhibitory potency of micropeptides is governed by their relative binding affinities to SERCA. This allows for finely tuned modulation of Ca²⁺ handling in different tissues based on differential expressions of micropeptide species. Understanding the contribution of each micropeptide to SERCA regulation may reveal novel strategies for therapeutic intervention in conditions where calcium dysregulation plays a role, such as heart disease, vascular disease, or neurodegenerative disorders.