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

With the use of a [H-3]ryanodine binding assay, the modulation of skeletal muscle ryanodine receptor (RyRl) by Zn2+ was investigated. In the presence of 100 mu M free Ca2+ concentration ([Ca2+](f)) as activator, the equilibri um [H-3]ryanodine binding to heavy sarcoplasmic reticulum vesicles was biph asically modulated by Zn2+. The binding was increased by a free Zn2+ concen tration ([Zn2+](f)) of less than 1 mu M; a peak binding, approx. 140 % of t he control (without added Zn2+) was obtained at 0.3 mu M [Zn2+](f). An inhi bitory effect of Zn2+ became obvious with a [Zn2+](f) of more than 1 mu M; the [Zn2+](f) for producing half inhibition was 2.7 +/- 0.5 mu M (mean +/- S.D.). Scatchard analysis indicated that the increase in the binding induce d by low [Zn2+](f) was due to a decrease in K-d, whereas both an increase i n K-d and a possible decrease in B-max were responsible for the decrease in binding induced by high [Zn2+](f). The binding in the presence of micro-mo lar [Zn2+](f) showed a biphasic time course. In the presence of 3 mu M [Zn2 +](f), after reaching a peak with an increased rate of initial binding, the binding gradually declined. The decline phase could be prevented by decrea sing [Zn2+](f) to 0.5 mu M or by adding 2mM dithiothreitol, a thiol-reducin g agent. The [Ca2+](f) dependence of binding was changed significantly by Z n2+, whereas Ca2+ had no clear effect on the [Zn2+](f) dependence of bindin g. Moreover, some interactions were found in the effects between Zn2+ and o ther RyR1 modulators. It is indicated that Zn2+ can modulate the activation sites and inactivation sites for Ca2+ on RyR1. The physiological significa nce of the effects of Zn2+ on ryanodine binding is discussed.