CHARACTERIZATION OF MULTIPLE [H-3] RYANODINE BINDING-SITES ON THE CA-2+ RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM FROM SKELETAL AND CARDIAC-MUSCLE - EVIDENCE FOR A SEQUENTIAL MECHANISM IN RYANODINE ACTION

Kinetic and equilibrium measurements of [H-3[ryanodine binding to the Ca2+ release channel of rabbit skeletal and rat cardiac sarcoplasmic reticulum (SR) are examined to ascertain the nature of cooperative interactions among high and low affinity binding sites and to quantitate their distribution. Equilibrium studies reveal affinities of 1-4 nM for the highest affinity binding site and of 30-50 nM, 500-800 nM, and 2-4-mu-M for the lower affinity sites in both preparations, with Hill coefficients of significantly < 1, and initial rates of association and dissociation increase with increasing concentrations of ryanodine. SR vesicles are actively loaded in the presence of pyrophosphate, and fluctuations in extravesicular Ca2+ are measured by the absorbance change of antipyrylazo III. The data demonstrate a biphasic, time- and concentration-dependent action of ryanodine on the release of Ca2+, with an initial activation and a subsequent inactivation phase. Kinetic analysis of the activation of Ca2+ release by ryanodine, in consonance with the binding data, demonstrates the existence of multiple binding sites for the alkaloid on the channel complex, with nanomolar to micromolar affinities. Based on the present findings obtained by receptor binding analysis and Ca2+ transport measurements, we suggest a model that describes four, most plausibly negatively cooperative, binding sites on the Ca2+ release channel. Occupation of ryanodine binding sites produces sequential activation followed by inactivation of the SR channel, revealing the strong possibility of an irreversible uncoupling of the native function of the receptor/channel complex by high concentrations of ryanodine. A model relating ryanodine receptor occupancy with SR Ca2+ release stresses two important new findings regarding the interaction of ryanodine with its receptor. First, ryanodine binds to four sites on the oligomeric channel complex with decreasing affinities, which can be best described by allosteric negative cooperativity. Second, binding of ryanodine to its receptor activates the Ca2+ release channel in a concentration-dependent and saturable manner in the range of 20 nM to 1 mM and produces a kinetically limited and sequential inactivation of the Ca2+ channel, with the concomitant attainment of full negative cooperativity. The results presented suggest that driving of the complex toward full negative cooperativity with high concentrations of ryanodine promotes a long-lived conformational state in which ryanodine is physically occluded and hindered from free diffusion from its binding site.