Ryanodine induces persistent inactivation of the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.
Junctional sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were pretreated with 0.1-500 microM ryanodine under equilibrium conditions optimal for receptor binding, followed by the removal of bound alkaloid by several washes in Ca(2+)- and ryanodine-free buffer. Pretreatment with > 100 nM ryanodine results in a concentration-dependent decrease in the Bmax of the high affinity sites and a complete loss of measurable low affinity binding sites that persist for > 48 hr. Quantitative analysis of residual ryanodine using three different methods demonstrates that the inhibition is not the result of residual ryanodine bound to its receptor. Ca2+ transport measurements made with antipyrylazo III show that actively loaded ryanodine-pretreated SR exhibits a persistent insensitivity to ryanodine- and daunomycin-induced Ca2+ release that is not seen with washed control vesicles. Lipid bilayer membranes fused with SR vesicles exhibit rapidly fluctuating single-channel events with a conductance of 468 pS in asymmetric CsCl solutions. Ryanodine (10 microM) produces a unidirectional transition to a slowly fluctuating half-conductance state that is not reversed by perfusion of the bilayer with Ca(2+)-free buffer and subsequent addition of dithiothreitol. However, dithiothreitol added in the ryanodine pretreatment medium offers marked protection against ryanodine-induced loss of binding sites and allows complete restoration of native gating behavior of single channels in bilayer lipid membrane. Using three different experimental approaches, the data demonstrate that the alkaloid at micromolar concentration persistently alters SR Ca2+ release channel function, perhaps by uncoupling four negatively cooperative binding sites. The oxidation of critical receptor thiols is implicated in the process.
Zimányi
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I
Department of Veterinary Pharmacology and Toxicology, University of California, Davis 95616.
Buck
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E
Abramson
J J
JJ
Mack
M M
MM
Pessah
I N
IN
D000818:Animals; D002121:Calcium Channel Blockers; D008051:Lipid Bilayers; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002121:Calcium Channel Blockers; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D013438:Sulfhydryl Compounds; D012433:Ryanodine