Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diap hragm by immunoaffinity chromatography using a specific antibody. The purif ied receptor was free from 12-kDa FK506-binding protein, although it retain ed the ability to bind 12-kDa FK506-binding protein. Negatively stained ima ges of RyR3 show a characteristic rectangular structure that was indistingu ishable 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 th e 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]r yanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid b ilayers, RyR3 displayed cation channel activity that was modulated by sever al ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent wit h [H-3]ryanodine binding activity. RyR3 showed a slightly larger unit condu ctance and a longer mean open time than RyR1, Whereas RyR1 showed two class es of channel activity with distinct open probabilities (P-o), RyR3 display ed a homogeneous and steeply Ca2+-dependent activity with P-o similar to 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidi zing 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.