FRET-Based RyR-Selective Detection Reveals No Significant Competition between CaM and S100A1 Binding to RyRs

Abstract

Using fluorescence resonance energy transfer (FRET), we directly tested the hypothesis that S100A1 competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. This hypothesis is largely based on competition assays using isolated sarcoplasmic reticulum (SR) membranes from skeletal muscle and evidence that S100A1 binds to peptides corresponding to one of the proposed CaM binding domains on RyR1. However, competition between S100A1 and CaM for RyR binding has not been directly detected. We targeted a donor-labeled FKBP12.6 (D-FKBP) to the cytoplasmic domain of RyR1 or RyR2 in SR vesicles isolated from pig skeletal or cardiac muscle. FRET was detected as a decrease of D-FKBP fluorescence in the presence of 100nM acceptor-labeled CaM (A-CaM) and used to index CaM binding to RyR. Upon pre-incubating SR with [S100A1] ranging from 0.01 to >100 μM, we found partial inhibition of FRET, with μM KI, for both skeletal and cardiac SR. By comparison, FRET was completely inhibited by unlabeled WT-CaM (KI ∼100nM), indicating that A-CaM and WT-CaM bind RyR with similar affinities and at the same site. Similar results were obtained using co-sedimentation assays conducted under similar experimental conditions, to detect competition between S100A1 and CaM binding to SR membranes. Taken together, these results indicate that CaM-RyR binding may not be significantly competed by S100A1 under normal physiologic conditions. Furthermore, structural analysis of FRET data suggests that S100A1 allosterically interacts with RyR-CaM binding. Initial results from a complementary FRET approach, using acceptor-labeled S100A1, further support the conclusion that S100A1 does not significantly compete with CaM-RyR binding in skeletal or cardiac muscle.