Molecular identification of the ryanodine receptor Ca2+ sensor.
We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR.
Chen
S R
SR
Cardiovascular Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada. swchen@acs.ucalgary.ca
Ebisawa
K
K
Li
X
X
Zhang
L
L
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence / Q000235:genetics; D004594:Electrophysiology; D005453:Fluorescence; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014162:Transfection / Q000235:genetics; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D014966:Xanthenes; C059715:Fluo-3; D000001:Calcimycin; D002110:Caffeine; D002118:Calcium