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

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 K(d), whereas both an increase in 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 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.