Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

Abstract

The ATP-dependent ion pump SERCA sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, this transporter is tightly controlled by physical interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. The distinct functions of these peptides may relate to their reciprocal preferences for SERCA binding. While SERCA binds PLB more avidly at low cytoplasmic Ca2+, it binds DWORF better at high Ca2+. In the present study, we determined that this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate conformations that the pump samples during the Ca2+ transport cycle. The results suggest a mechanistic basis for inhibitory and stimulatory micropeptide function. In addition, fluorescence resonance energy transfer (FRET) measurements revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. The data suggest Ca2+-dependent dynamic exchange of inhibitory and stimulatory micropeptides from SERCA during the cardiac cycle. Together, these mechanisms provide beat-to-beat modulation of cardiac Ca2+ handling and contribute to the hearts adaptation to the increased physiological demands of exercise. O_TEXTBOXSignificance Interactions between SERCA and its regulatory micropeptides modulate cardiac performance. A previous study demonstrated that while the SERCA inhibitor, phospholamban, loses affinity during intracellular Ca2+ elevations, its competitor DWORF stimulates SERCA and binds better at elevated Ca2+. Here, we found this Ca2+-dependent difference in affinity is driven by preferential micropeptide binding to different SERCA conformations sampled during its enzymatic cycle. Tight binding to different conformations of the pump may underly the distinct mechanistic functions of these regulators. Lower affinity during alternating phases of the cardiac cycle may drive dynamic exchange of inhibitory and stimulatory micropeptides during contraction and relaxation. Our results reveal mechanisms that modulate cardiac Ca2+ handling on a beat-to-beat basis and help the heart adapt to exercise. C_TEXTBOX