The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain.
A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.
Zhu
Xinsheng
X
Department of Physiology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706, USA.
Ghanta
Jyothi
J
Walker
Jeffrey W
JW
Allen
Paul D
PD
Valdivia
Héctor H
HH
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013552:Swine
D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine