FRET Detection of CaM-RyR2 Binding Modulation by S100A1

Abstract

Using fluorescence resonance energy transfer (FRET), we are testing the hypothesis that S100A1 competes with calmodulin (CaM) for binding to cardiac ryanodine receptors (RyR2). In isolated pig cardiac sarcoplasmic reticulum (SR) vesicles, we targeted a donor-labeled FKBP (D-FKBP) to the RyR2 cytosolic headpiece. We then detected FRET as a decrease of donor fluorescence in the presence of CaM labeled with acceptor within the N- or C-lobe (denoted AN-CaM and AC-CaM, respectively), thus directly and specifically indexing CaM binding in the proximity of D-FKBP. FRET between D-FKBP and A-CaMs (100 nM) was completely inhibited by unlabeled WT-CaM, with KI ≈ 100 nM, indicating that WT-CaM and A-CaMs compete with similar affinities for the same RyR2 binding site. However, S100A1 (ranging from 0.1 to 30 μM) had no significant effect on FRET when cardiac SR was concurrently incubated with S100A1 and AN-CaM. Upon sequentially incubating the SR with S100A1 first, then with AN-CaM, we found partial inhibition of FRET, but with KI ≈ 30 μM S100A1. This effect is more pronounced in nM Ca2+ (versus μM Ca2+). Intriguingly, FRET between D-FKBP and AC-CaM was not significantly affected by S100A1. S100A1 lowered maximum FRET for AN-CaM but did not significantly change its binding affinity. This suggests that S100A1 allosterically interacts with RyR-CaM binding, rather than directly competing for the same binding site as CaM. We are currently developing a complementary FRET approach, using acceptor-labeled S100A1, to specifically resolve the S100A1 binding to RyR. Ultimately, we aim to elucidate the interplay between S100A1 and CaM binding to RyR.