The N-Termini of Sarcolipin and Phospholamban Play an Important Role in Distinct Regulation of Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) Function

Abstract

The Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) is responsible for intracellular Ca2+ homeostasis in muscle and non-muscle cells. SERCA activity in muscle can be regulated by Phospholamban (PLB), an affinity modulator, and Sarcolipin (SLN), an uncoupler. While PLB gets dislodged from Ca2+-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca2+ transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. We demonstrated that deletion of SLN N-terminal residues 2ERSTQ leads to loss of uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Further, Molecular Dynamics simulations of SLN and SERCA interaction showed that the deletion of SLN N-terminus introduces structural rearrangement of SERCA residues. Interestingly, transfer of the PLB cytosolic domain to SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. We also found that swapping the PLB N-terminus with the SLN N-terminus caused the resulting chimera to acquire SLN-like function. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however the resulting chimera acquires a function different from the parent molecule. Importantly, our studies highlight that the N-termini of SLN and PLB affect their respective unique function by proper positioning of the TM domain along with C-terminus on SERCA.