Glutathione Modulates Ryanodine Receptor from Skeletal Muscle Sarcoplasmic Reticulum

Abstract

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. In muscle cells, cytosolic Ca 2ϩ levels are regulated by the intramuscular organelle, the sarcoplasmic reticulum (SR) 1 (1-3). Following the arrival of an action potential at the surface membrane and subsequent depolarization of the transverse tubule, the SR releases its lumenal store of Ca 2ϩ through the Ca 2ϩ release channel (CRC)/ryanodine receptor (RyR), thus triggering the contraction process. The interaction between the action potential at the transverse tubule and the release of Ca 2ϩ from the SR has been termed excitation-contraction coupling (ECC). In skeletal muscle, the molecular mechanism underlying ECC has remained unclear. Following excitation, resting Ca 2ϩ levels are reestablished through the active transport of the Ca 2ϩ back into the lumen of the SR by the Ca release by a large class of non-thiol channel stimulators, a high molecular weight complex was formed. The addition of channel inhibitors resulted in the reduction of the disulfides formed, the dissociation of key SR proteins and the exposure of hyperreactive thiols (14,15). Based on the results derived from fluorescence assays, ion flux measurements, single-channel experiments, and SDS-gel electrophoresis, the authors concluded that thiol oxidation-reduction chemistry plays a critical role in the channel gating of the SR CRC⅐RyR complex.Endogenous and exogenous redox agents have been observed to have profound effects on a wide range of ion channel systems. In addition to the SR Ca 2ϩ release channel, ion channels as varied as excitatory amino acid receptors, inositol 1,4,5-triphosphate (IP 3 )-gated Ca 2ϩ release channels, and even certain K ϩ channels have been demonstrated to be modulated by redox agents. For example, the N-methyl-D-aspartate-sensitive excitatory amino acid receptor has been shown to be modulated by both thiol oxidants and reductants (16).Glutathione is one of the most abundant low molecular weight peptides in eukaryotic cells and the most prevalent intracellular thiol. Depending on the cell type, glutathione levels have been estimated to range from 1 to 10 mM (17, 18). In the cell, glutathione acts as both a reducing agent and an antioxidant. Among its many physiological roles, glutathione * This work was supported by a grant from the American Heart Association Oregon Affiliate (to J. J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom all correspondence should be addressed: Physics Dept., Portland State University, P. O. Box 751, Portland, 1 The abbreviations used are: SR, sarcoplasmic reticulum; CRC, Ca 2ϩ release channels; RyR, ryanodine receptor; RyR 1 , ryanodine receptor (skeletal isoform); ECC, excitation-contraction coupling; CPM, 7-diethylamino-3-(4Ј-maleimidylpheny...

Biphasic modulation of ryanodine binding to sarcoplasmic reticulum vesicles of skeletal muscle by Zn2+ ions

“…The Scatchard analysis was based on a one-site model [5]. From the plot of the ratio of bound to free ryanodine (B\F ) against B, K d (the equilibrium binding constant) and B max (the maximal number of ryanodine-binding sites) were estimated from the equation B\F l (B max kB)\K d .…”

“…It has been shown that the function of RyR1s can be modulated by various endogenous and exogenous factors, including caffeine, Mg# + and adenine nucleotide [2][3][4][5]. It is thought that caffeine increases the apparent affinity of the activation site for Ca# + , whereas Mg# + inhibits ryanodine binding by competing with Ca# + for the Ca# + activation site [3,4].…”

“…Unless indicated otherwise, [$H]ryanodine binding assays were performed as described elsewhere [5], with some modifications. …”

“…It has been shown that the function of RyR1s can be regulated by various endogenous and exogenous factors, including several univalent and bivalent ions [2][3][4][5]. Among the bivalent cations, the effects of Ca# + and Mg# + are well known [3,4].…”

“…As a convenient method, a [$H]ryanodine binding assay has been used to investigate the effect of various agents on the function of RyR1s [2][3][4][5]. A close correlation between ryanodine binding and the gating state of RyR1s has been established for many ligands of RyR1s [2], although there are a few exceptions [4,8].…”