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

Abstract

With the use of a [3H]ryanodine binding assay, the modulation of skeletal muscle ryanodine receptor (RyR1) by Zn2+ was investigated. In the presence of 100 μM free Ca2+ concentration ([Ca2+]f) as activator, the equilibrium [3H]ryanodine binding to heavy sarcoplasmic reticulum vesicles was biphasically modulated by Zn2+. The binding was increased by a free Zn2+ concentration ([Zn2+]f) of less than 1 μM; a peak binding, approx. 140% of the control (without added Zn2+) was obtained at 0.3 μM [Zn2+]f. An inhibitory effect of Zn2+ became obvious with a [Zn2+]f of more than 1 μM; the [Zn2+]f for producing half inhibition was 2.7±0.5 μM (mean±S.D.). Scatchard analysis indicated that the increase in the binding induced by low [Zn2+]f was due to a decrease in Kd, whereas both an increase in Kd and a possible decrease in Bmax were responsible for the decrease in binding induced by high [Zn2+]f. The binding in the presence of micromolar [Zn2+]f showed a biphasic time course. In the presence of 3 μM [Zn2+]f, after reaching a peak with an increased rate of initial binding, the binding gradually declined. The decline phase could be prevented by decreasing [Zn2+]f to 0.5 μM or by adding 2 mM dithiothreitol, a thiol-reducing agent. The [Ca2+]f dependence of binding was changed significantly by Zn2+, whereas Ca2+ had no clear effect on the [Zn2+]f dependence of binding. Moreover, some interactions were found in the effects between Zn2+ and other RyR1 modulators. It is indicated that Zn2+ can modulate the activation sites and inactivation sites for Ca2+ on RyR1. The physiological significance of the effects of Zn2+ on ryanodine binding is discussed.