Ryanodine stabilizes multiple conformational states of the skeletal muscle calcium release channel

Edmond Buck, Ildiko Zimanyi, Jonathan J. Abramson, Isaac N Pessah

Research output: Contribution to journalArticlepeer-review

140 Scopus citations

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 μM 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 Ca2+-induced Ca2+ release from actively loaded SR vesicles. Ryanodine ≥50 nM stabilizes the channel in a 234-pS subconductance which is not readily reversible. RY (≥70 μM) produces a unidirectional transition from the 1/2 to a 1/4 conductance fluctuation, whereas RY ≥200 μM 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.

Original languageEnglish (US)
Pages (from-to)23560-23567
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number33
StatePublished - Nov 25 1992

ASJC Scopus subject areas

  • Biochemistry

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