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 RS RS homotetramer had a single class of high affinity [H-3]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 [H-3]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 (P-o), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with P-o similar to 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 [H-3]ryanodine binding and channel activity.