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The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the "regulins". Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA-formed by transmembrane helices M2, M6, and M9-can result in distinct functional outcomes.

The sarcoplasmic reticulum Ca 2+ -ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)