Regulation of SERCA1 in Skeletal Muscle by sAnk1: Binding to Phospholamban and the Role of sAnk1’s Cytoplasmic Domain

Sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase1 (SERCA1) is responsible for the clearance of cytosolic Ca 2+ in skeletal muscle, and its regulation has been intensively studied. Small ankyrin 1 (sAnk1, Ank1.5), a 17 kDa muscle-specific isoform of ANK1, binds to SERCA1 directly via both its transmembrane and cytoplasmic domains and inhibits SERCA1’s ATPase activity. We characterized the interaction between the cytoplasmic domain of sAnk1 (sAnk1(29-155)) and SERCA1. The binding affinity was 444 nM by blot overlay, about 7-fold weaker than the binding of sAnk1(29-155) to obscurin, a giant protein of the muscle cytoskeleton. Site-directed mutagenesis identified K38, H39, and H41, in the juxtamembrane region, as essential for binding to SERCA1 but not to obscurin. Residues R64-K73, which contribute to obscurin binding, are also required for binding to SERCA1, but only the hydrophobic residues in this sequence are required, not the positively charged residues necessary for obscurin binding. Circular dichroism analysis of sAnk1(29-155) indicates that most mutants we studied show significant structural changes, with the exception of K38A/H39A/H41A. Although the cytoplasmic domain of sAnk1 does not inhibit SERCA1’s Ca 2+ -ATPase activity, with or without mutations in the juxtamembrane sequence, the inhibitory activity of full-length sAnk1 requires the WT juxtamembrane sequence. We used these data to model sAnk1 and the sAnk1-SERCA1 complex. We showed previously that sAnk1 binds to sarcolipin (SLN), a 31-amino acid peptide inhibitor of SERCA1, forming a three-way complex with SERCA1. Notably, the presence of sAnk1 ablates SLN’s inhibitory effect on SERCA1. We have now examined the binding of sAnk1 to phospholamban (PLN). We found that sAnk1 binds directly to PLN and also forms a three-way complex with SERCA1. This interaction does not ablate PLN’s inhibition of SERCA1 activity, however. Our results are consistent with a model in which, in forming a three-way complex, PLN binds to SERCA1 first, followed by binding to sAnk1. Our results suggest that, in addition to its transmembrane domain, sAnk1 uses its juxtamembrane sequence and perhaps part of its obscurin binding site to bind to SERCA1, and that this binding contributes to their robust association in situ, as well as regulation of SERCA1’s activity. Our studies with PLN suggest that the interactions of PLN, SLN, and sAnk1 with SERCA1, either alone or in pairs, are distinct and have different effects on SERCA1’s enzymatic activity. NIH RO1 AR077106 to R.J. Bloch and A. Kontrogianni-Konstantopoulos. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.