Molecular basis of calmodulin binding to cardiac muscle Ca2+ release channel (ryanodine receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at > 1 muM Ca2+ but activates RyR1 and inhibits RyR2 at < 1 μM Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [S-35] CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 μM. Five RyR2 single or double mutants in the CaM binding region ( W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [ 35S] CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [ 35S] CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.