Effect of Tempreature on [3H] Ryanodine Binding to Sarcoplasmic Reticulum from Bullfrog Skeletal Muscle1

Abstract

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 sarcoplasmic 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 beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees 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 degrees 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 degrees 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 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Similar Ca2+ dependences of [3H]ryanodine binding to α‐ and β‐ryanodine receptors purified from bullfrog skeletal muscle in an isotonic medium

“…This clearly indicates that the Ca 2+ sensitivity of ~-RyR was enhanced in 1 M NaCI medium, while that of a-RyR remained unchanged. The results including those of Figs SR vesicles under similar conditions [16]. One mM AM-POPCP markedly enhanced the binding (by a factor of more than 10 at pCa 4.5 for ct-or 13-RyR) with minor leftward shift of the pCa-binding curve in 0.17 M NaC1 medium, while the stimulation was less marked in 1 M NaCI medium.…”

“…Caffeine sensitized [3H]ryanodine binding to Ca 2+ activation with only slight increase in the maximal binding, in contrast to the effect of AMPOPCP. The effect of caffeine was weaker in 0.17 M NaCl-medium than that in 1 M NaCl-medium or that with SR vesicles in 0.17 M and 1 M NaC1 medium [12,16]. The combined effect of AMPOPCP and caffeine appeared potentiating in 0.17 M NaCI medium, while it was additive in 1 M NaC1 medium.…”

Calcium-Induced Calcium Release in Skeletal Muscle

“…This was also first shown in skinned fibers and then in FSR (50,54,55,110,118,168,171,181,182,191). The order of potency was ATP ϳ adenosine 5Ј-(␤,␥-methylene)triphosphate (AMPPCP) Ͼ ADP ϳ cAMP Ͼ AMP Ͼ adenine Ͼ adenosine (54,118,168,181 are markedly increased by ATP and its analogs in the same manner (32,96,143,144,182,199,213). An interesting finding was that adenine and adenosine inhibit CICR in the presence of ATP.…”

“…(50,59,182) and then in fragmented SR (FSR) (122,124,168,191). A similar biphasic dependence on cis (cytoplasmic) Ca 2ϩ is shown by the open probability of the purified Ca 2ϩ release channel RyR, incorporated into a lipid bilayer, and by [ 3 H]ryanodine binding to RyR (19,26,31,113,182,197,199,212,213,236), consistent with the notion that CICR takes place via the RyR. The biphasic effect of Ca 2ϩ suggests the presence of two Ca 2ϩ receptor sites on RyRs: a high-affinity activating site (CICR A-site) and a lowaffinity inhibitory site (CICR I-site) (169,198).…”

“…An interesting finding was that adenine and adenosine inhibit CICR in the presence of ATP. This inhibition occurs because the strong potentiation of CICR by ATP is replaced by the much weaker potentiation by adenine or adenosine (42,52,110,150,169 (48,50,124,168,171,182,191,199,213,223 (50,199). In mammalian skeletal and cardiac muscle, however, a high concentration of caffeine induces Ca 2ϩ release via RyR in the virtual absence of Ca 2ϩ (0.08 -2 nM), provided that Mg 2ϩ is absent (223,243).…”