Abstract
Nanomolar to micromolar ryanodine alters the gating kinetics of the Ca2+ release channel from skeletal sarcoplasmic reticulum (SR) fused with bilayer lipid membranes (BLM). In the presence of asymmetric CsCl and 100 microM CaCl2 cis, ryanodine (RY) (5-40 nM) activates the channel, increasing the open probability (po; maximum 300% of control) without changing unitary conductance (468 picosiemens (pS)). Statistical analyses of gating kinetics reveal that open and closed dwell times exhibit biexponential distributions and are significantly modified by nanomolar RY. Altered channel gating kinetics with low nanomolar RY is fully reversible and correlates well with binding kinetics of nanomolar [3H]RY with its high affinity site (Kd1 = 0.7 nM) under identical experimental conditions. RY (20-50 nM) induces occasional 1/2 conductance fluctuations which correlate with [3H]RY binding to a second site having lower affinity (Kd2 = 23 nM). RY (5-50 nM) in the presence of 500 mM CsCl significantly enhances Ca(2+)-induced Ca2+ release from actively loaded SR vesicles. Ryanodine > or = 50 nM stabilizes the channel in a 234-pS subconductance which is not readily reversible. RY (> or = 70 microM) produces a unidirectional transition from the 1/2 to a 1/4 conductance fluctuation, whereas RY > or = 200 microM causes complete closure of the channel. The RY required for stabilizing 1/4 conductance transitions and channel closure do not quantitatively correlate with [3H]RY equilibrium binding constants and is attributed to significant reduction in association kinetics with > 200 nM [3H]RY in the presence of 500 mM CsCl. These results demonstrate that RY stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting RY effector sites on the channel protein.
Highlights
Nanomolar to micromolar ryanodine alters the gat- tion coupling of striated muscle
Receptor Binding Studies in thePresence of CsC1-The multiplicity of [3H]ryanodine binding sites in skeletal sarcoplasmic reticulum (SR) membranes is evident when equilibrium (4.5-h incubation) binding is performed within two discriminating concentration ranges of radioligand at 25 "C in the presence of 500 mM CsCl and 100 PM CaC12
During excitation-contraction coupling in striated muscle, to alter single channel-gating behavior and the quantitative the release of Ca2+from SR appears to be mediated by a dose-response relationship in BLM experiments
Summary
Preparation of SR Vesicles-Purified SR vesicles from rabbit skeletal musclewere prepared by the method of Saitoet al. [18] for receptor analysis and Ca2+transport measurements or by the method of MacLennan [19] for BLM experiments. Measurement of L3H]RyanodineBinding-Specific equilibrium binding of [3H]ryanodine (95or 60Ci/mmol, with or 97.2%purity, respectively; Du Pont-New England Nuclear) was performed under conditions identical to those used for obtaining single channel measurements in planar bilayer (500 mM csc pM CaC12, 5 mM HEPES, pH 7.2, a t 25 "C). To determine the binding constants (Kd, BmaJof the high and low affinity binding sites, equilibrium experiments (4.5-h incubation) were performed ina1-ml volume in the presence of 0.5-500 or 50-5050 nM [3H]ryanodinein a final volume of 1 ml, respectively. 30 pg of SR vesicles wereincubated in duplicate for various times with 1nM [3H]lyan~din(eforthe high affinity state) or 50, 250, 500,1000,or3000 nM [3H]ryanodine (for low affinity states) in a final volume of 1ml of 500 mM CsC1, 5 mM HEPES, pH.
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