Effect of Temperature on [³H] Ryanodine Binding to Sarcoplasmic Reticulum from Bullfrog Skeletal Muscle

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It has been clarified that ryanodine binds to Ca2⁺-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarco-plasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2⁺ is indispensable for ryanodine binding, Ca2⁺ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2⁺, caffeine and/or β, γ-methylene adenosine triphosphate (AMPOPCP) are necessary. [³H]Ryanodine binding at 25°C closely paralleled the Ca release activity in respect of the Ca²⁺- dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0°C, the rate of ryanodine binding decreased, Q₁₀, being about 3 on average. The affinity for ryanodine was reduced to about half that at 25°C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca²⁺ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0°C. These results indicate that the open state of Ca²⁺ -induced Ca release channels is requisite for ryanodine binding and that ryanodine binding to the open state is a distinct process. The physicochemical aspects of ryanodine binding are also discussed.