Antibodies as probes for Ca2+ activation sites in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.
In earlier studies (Chen, S. R. W., Zhang, L., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 23318-23326), an amino acid sequence, designated 13c2, lying between amino acid residues 4478 and 4512 in the skeletal muscle ryanodine receptor was shown, through the use of a polyclonal antibody, to be involved in Ca(2+)-induced Ca2+ release. In the present study, an immobilized synthetic peptide, PEPEPEPEPE, corresponding to part of the predicted high affinity Ca2+ binding site between residues 4489 and 4499, was used to purify specific antibodies from an anti-13c2 rabbit antiserum. The effect of this affinity-purified, anti-peptide (anti-13cp1) antibody on Ca2+ release channel function was then characterized using single channel recordings across planar lipid bilayers. The anti-peptide antibody inhibited Ca(2+)- or caffeine-activated channel activities without closing the channel but did not diminish ATP-activated channel activity. The addition of ATP reversed the inhibition of the Ca(2+)- or caffeine-activated channel by the antibody, and the antibody-bound, ATP-activated channel was further modulated by Mg2+, ryanodine, and ruthenium red. The major epitopes in the anti-13c2 antibody, previously shown to activate the Ca2+ release channel by increasing the Ca2+ sensitivity of the channel, did not lie in the PE repeat. These results suggest that the PE repeat sequence forms a site involved in the Ca2+ activation pathway.
Chen
S R
SR
Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Ontario, Canada.
Zhang
L
L
MacLennan
D H
DH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D000939:Epitopes; D015640:Ion Channel Gating / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D000906:Antibodies; D015220:Calcium Channels; D000939:Epitopes; D009124:Muscle Proteins; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium