Identification of calmodulin-, Ca(2+)-, and ruthenium red-binding domains in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.
cDNAs encoding trpE fusion proteins containing fragments of the skeletal muscle Ca2+ release channel (ryanodine receptor) were expressed in bacteria. The fusion proteins, which covered about 90% of the linear sequence of the ryanodine receptor, were used to identify calmodulin- (CaM), Ca(2+)-, and ruthenium red-binding regions in the ryanodine receptor through the use of 125I-CaM, 45Ca2+, and ruthenium red overlay procedures. Six Ca(2+)-dependent CaM-binding domains were detected in the skeletal muscle ryanodine receptor. Strong CaM-binding domains were localized in regions 6, 11, 12, and 13, in subregions 6b, 11b, and 13b, and in short sequences 6b3, 11b1, and 13b2, lying between amino acid residues 2063 and 2091, 3611 and 3642, and 4303 and 4328. Weaker CaM-binding domains were localized in regions 4, 9, and 10 and in subregions 4b, 9b, and 10a, lying between residues 921 and 1173, 2804 and 2930, and 2961 and 3084. Most of these CaM-binding domains encompassed all or part of previously predicted CaM-binding sites. Strong 45Ca(2+)- and ruthenium red-binding sites domains were localized in the NH2- and COOH-terminal regions of the ryanodine receptor and in regions 6, 12, and 13. The 45Ca(2+- and ruthenium red-binding sites in regions 6 and 12 were localized in subregions 6b and 12b, lying between residues 1861-2094 and 3657-3776. These data together with earlier studies (Chen, S. R. W., Zhang, L., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 23318-23326), show that strong CaM-, Ca(2+)-, and ruthenium red-binding domains are colocalized in the skeletal muscle ryanodine receptor.
Chen
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SR
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
MacLennan
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DH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D017931:DNA Primers; D004533:Egtazic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012430:Ruthenium Red / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D015220:Calcium Channels; D002147:Calmodulin; D017931:DNA Primers; D009124:Muscle Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D004533:Egtazic Acid; D002118:Calcium