Effects Of Zinc Ions And Divalent Cation's Chelators On Ryanodine Receptors Of Skeletal And Cardiac Muscle

Using [3H]ryanodine binding assay, the effects of Zn2+ and divalent cation's chelators on ryanodine receptors(RyRs) of skeletal and cardiac muscle cells were investigated. The [3H]ryanodine binding to type I ryanodine receptor (RyR1) on heavy sarcoplasmic reticulum vesicles of rabbit skeletal muscle was biphasically modulated by Zn2+ in the presence of 100 (M [Ca2+]f. The binding was increased by Zn2+ when [Zn2+]f was less than 1 (M, while the inhibitory effect of Zn2+ became obvious when the [Zn2+]f was over 1 (M, with an IC50/ZnI was 2.7(0.5 (M (Mean(S.D.). The binding in the presence of inhibitory [Zn2+]f also showed a biphasic time course. It is indicated that two types of Zn2+ binding sites are present on RyR1s: activation sites and inhibitory sites. [Ca2+]f dependence of ryanodine binding was significantly changed by Zn2+, while Ca2+ had no clear effect on [Zn2+]f dependence of the binding. Moreover, some interactions in the effects on ryanoding binding were found between Zn2+ and RyR modulators. All of these results indicate that Zn2+ can modulate the activation sites and inactivation sites for Ca2+ on RyR1s. The biphasic effect of Zn2+ on RyR1s was antagonized by 2 mM dithiothreitol, a thiol-reducing agent, suggesting the involvement of RyR1s' thiols in the regulation of RyR1s by Zn2+ and the presence of inherent Zn2+ in RyR1sThe [3H]ryanodine binding to the type II ryanodine receptor (RyR2) on sarcoplasmic reticulum of calf cardiac muscle was monophasically inhibitedby [Zn2+]f , with an IC50/ZnI of 2.1(0.4 (M. The interactions of the effects of Zn2+ with other modulators of RyRs indicate that the inhibitory effect of Zn2+ was mostly mediated through inhibiting Ca2+ activation sites on RyR2s. Since the inhibitory effect on ryanodine binding of Zn2+ was not clearly changed by [Ca2+]f, the effect of Zn2+ may not be due to competition between Zn2+ and Ca2+ for Ca2+ activation sites but probably be due to indirect action. Unlike that on RyR1s, the inhibitory effect of Zn2+ on RyR2s could not be antagonized by 2 mM dithiothreitol. We also found for the first time that, EGTA and TPEN, divalent cation's chelators used for keeping constant [Ca2+]f in ryanodine binding assays, inhibited ryanodine binding to RyR1s and RyR2s. The IC50-EGTA and IC50-TPEN were 3.4(0.3 mM and 50 ?M. The inhibitory potency of these chelators on ryanodine inding is consistent with their affinity with Zn2+. Interestingly, the inhibitory effect of 5 mM EGTA on ryanodine binding could be prevented by added Zn2+. But, the depression effect induced by 100 ?M TPEN could be partially recovered by Zn2+, only in the presence of 2 mM GSH, a thiol-reducing agent. Our results show that RyRs can be regulated by Zn2+. Moreover, that RyR1 and RyR2 were differentially affected by Zn2+ indicates that the effect of Zn2+ on RyRs may be isoform-dependent. The present results also suggest the presence of inherent Zn2+ in RyRs, which is important for normal functioning of RyRs. Moreover, the result about the inhibitory effect on ryanodine binding of concentrated chelators indicates that using proper concentrations of chelators is essential in ryanodine binding assays.