Molecular basis of calmodulin binding to cardiac muscle Ca(2+) 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 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm 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 [35S]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 microm. 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.
Yamaguchi
Naohiro
N
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Xu
Le
L
Pasek
Daniel A
DA
Evans
Kelly E
KE
Meissner
Gerhard
G
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D009154:Mutation; D009206:Myocardium / Q000737:chemistry*; D011485:Protein Binding; D020033:Protein Isoforms / Q000819:agonists / Q000037:antagonists & inhibitors; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011849:Radioactive Tracers; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D014316:Tritium
D002147:Calmodulin; D020033:Protein Isoforms; D011849:Radioactive Tracers; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D002118:Calcium