PHOTOAFFINITY-LABELING OF THE RYANODINE RECEPTOR CA2+ RELEASE CHANNELWITH AN AZIDO DERIVATIVE OF RYANODINE

Ryanodine receptors/Ca2+ release channels play an important role in re gulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work descr ibed here, we characterize the interaction of a tritium- labeled, phot oactivable derivative of ryanodine (H-3-labeled 10 O-[3-(4-azidobenzam ido)propionyl]ryanodine ([H-3]ABRy)) with the ryanodine receptor of sk eletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in ske letal muscle triads. Furthermore, competition binding assays of [[H-3] ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this az ido derivative of ryanodine is able to specifically displace [H-3]ryan odine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCI on [H-3]ABRy binding in triad membranes shows a similar modula tion of binding to that seen in these membranes with [H-3]ryanodine. P hotoaffinity labeling of triads with [H-3]ABRy resulted in specific an d covalent incorporation of [H-3]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the l abeled ryanodine receptor revealed a [H-3]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COO H-terminal of the receptor. These results demonstrate that the 76-kDa COOH- terminal tryptic fragment contains the high affinity binding sit e for ryanodine.