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

ABSTRACT

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