The Ca2+-ryanodine receptor complex is solubilized in functional form on treating sarcoplasmic reticulum (SR) vesicles from rabbit fast skeletal muscle with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (1 mg/mg protein) and 1 M NaCl at pH 7.1 by shaking for 30 min at 5°C. The heavy membrane preparations obtained from pyrophosphate homogenates frequently exhibit junctional feet and appear to be derived primarily from the terminal cisternae of the SR. The characteristics of [3H]ryanodine binding are similar for the soluble receptor and the heavy SR vesicles with respect to dependence on Ca2+, pharmacological specificity for inhibition by six ryanoids and ruthenium red, and lack of sensitivity to voltage-dependent Ca2+-channel blockers, inositol 1,4,5-trisphosphate, or doxorubicin. In contrast, the cation sensitivity is decreased on receptor solubilization. The soluble receptor is modulated by cyclic nucleotides and rapidly denatured at 50°C. Saturation experiments reveal a single class of receptors (K(d) = 9.6 nM), whereas kinetic measurements yield a calculated association constant of 5.5 x 106 min-1 M-1 and a dissociation constant of 5.7 x 10-4 min-1, suggesting that the [3H]ryanodine receptor complex ages with time to a state which is recalcitrant to dissociation. Sepharose chromatography shows that the receptor complex consists primarily of two protein fractions, one of apparent M(r) 150,000-300,000 and a second, the [3H]ryanodine binding component, of approximately M(r) 1.2 x 106. Preliminary analysis of the soluble receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals subunits of M(r) >200,00 and major bands of calsequestrin and Ca2+-transport ATPase. These findings indicate that [3H]ryanodine binds to the Ca2+-induced open state of the channel involved in the release of contractile Ca2+.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1986|
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