Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca(2+)-induced Ca2+ release.
In the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel. cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel. Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues). Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae. Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae. These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop 2.
Treves
S
S
Istituto di Patologia Generale, Università degli Studi di Ferrara, Italy.
Chiozzi
P
P
Zorzato
F
F
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000276:immunology / Q000502:physiology*; D002846:Chromatography, Affinity; D004262:DNA Restriction Enzymes; D000939:Epitopes / Q000276:immunology; D009124:Muscle Proteins / Q000737:chemistry* / Q000276:immunology / Q000502:physiology*; D009132:Muscles / Q000737:chemistry; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D019837:Ryanodine Receptor Calcium Release Channel
D000906:Antibodies; D015220:Calcium Channels; D000939:Epitopes; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D004262:DNA Restriction Enzymes; D002118:Calcium