Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.
We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (Po), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with Po approximately 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.
Murayama
T
T
Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
Oba
T
T
Katayama
E
E
Oyamada
H
H
Oguchi
K
K
Kobayashi
M
M
Otsuka
K
K
Ogawa
Y
Y
D000818:Animals; D000918:Antibody Specificity; D002110:Caffeine / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D003964:Diaphragm; D004553:Electric Conductivity; D020104:Immunophilins / Q000378:metabolism; D015640:Ion Channel Gating; D008274:Magnesium / Q000494:pharmacology; D016624:Negative Staining; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000276:immunology; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure; D012519:Sarcoplasmic Reticulum; D013439:Sulfhydryl Reagents; D022021:Tacrolimus Binding Proteins
D002413:Cations, Divalent; D010446:Peptide Fragments; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D013439:Sulfhydryl Reagents; D012433:Ryanodine; D002110:Caffeine; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D008274:Magnesium; D002118:Calcium