BPS2026 – Fluorescence lifetime spectroscopy reveals micropeptide regulation of SERCA homodimers

The sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) is responsible for pumping cytosolic calcium into the sarcoplasmic reticulum (SR), thus determining the rate of cardiac relaxation and the strength of the subsequent cardiac contraction. SERCA activity decreases in heart failure (HF), making the protein and its regulatory network a prominent therapeutic target. Here, we assessed the interaction of SERCA with its micropeptide regulatory partner phospholamban (PLB), an inhibitor, and dwarf open reading frame (DWORF), a stimulator of SERCA transport activity. We transfected HEK293 cells with OFP-labeled SERCA and mMaroon-labeled PLB or DWORF and quantified Förster resonance energy transfer (FRET) using time-correlated single photon counting (TCSPC). We generated fluorescence lifetime imaging microscopy images and performed extended analysis of single cells by parking the excitation laser on one location in the cell. Multi-exponential fitting of TCSPC decays revealed distinct populations of FRET donors. Overall, the data suggest the micropeptides interact with a dimeric complex of two SERCA pumps. This is consistent with a model in which functional coupling of SERCA pumps enhances calcium transport. Further studies of the physiological significance of SERCA homodimerization will utilize optogenetic approaches for spatiotemporal control SERCA-SERCA interactions. A better understanding of how SERCA is regulated may inform future therapeutic strategies to improve calcium handling in HF.