Characterization of a Ca2+ binding and regulatory site in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.
A region in the skeletal muscle ryanodine receptor between amino acids 4014 and 4765 was expressed as a trpE fusion protein. Overlay studies revealed that this region bound Ca2+ and ruthenium red, an indicator of Ca(2+)-binding sites. Ca2+ binding was mapped to subregion 13b between amino acids 4246 and 4377, encompassing a predicted high affinity Ca(2+)-binding site, and to subregion 13c between amino acids 4364 and 4529, encompassing two predicted high affinity Ca(2+)-binding sites. Ca2+ binding was then mapped to three shorter sequences, 22(13b1), 36(13c1), and 35(13c2), amino acids long, each encompassing one of the three predicted Ca(2+)-binding sites. Site-directed polyclonal antibodies were raised against these three short sequences and purified on antigen affinity columns. The antibody against sequence 13c2, lying between residues 4478 and 4512, specifically recognized both denatured and native forms of the ryanodine receptor, suggesting that at least part of the 35 amino acid sequence containing the Ca(2+)-binding site is surface-exposed. The affinity purified antibody increased the Ca2+ sensitivity of ryanodine receptor channels incorporated into planar lipid bilayers, resulting in increased open probability and opening time without altering channel conductance. The antibody-activated channel was still modulated by Ca2+, Mg2+, ATP, ryanodine, and ruthenium red. These observations suggest that sequence 13c2 may be involved in Ca(2+)-induced Ca2+ release.
Chen
S R
SR
Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Ontario, Canada.
Zhang
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L
MacLennan
D H
DH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology; D008051:Lipid Bilayers; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012430:Ruthenium Red / Q000378:metabolism / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D007473:Ion Channels; D008051:Lipid Bilayers; D009838:Oligodeoxyribonucleotides; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D012433:Ryanodine; D002118:Calcium