RAPID KINETICS OF MYOINOSITOL TRISPHOSPHATE BINDING AND DISSOCIATION IN CEREBELLAR MICROSOMES

Using sheep cerebellum microsomes adsorbed on a filter, we measured th e kinetics of [H-3]inositol 1,4,5-trisphosphate (InsP(3)) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [(3H)]InsP(3)-containing or InsP(3)-free media. At 20 deg rees C and pH 7.1, in a cytosol-Like medium containing MgCl2, the half -time for InsP(3) dissociation was as short as 125 ms. The receptor be haved as a simple target for binding of its ligand, with the rate cons tant for InsP(3) binding increasing Linearly with InsP(3) concentratio n. Various modulators of InsP(3) binding (KCl, NaCl, pH, Mg2+, and Ca2 +) mere found to affect the receptor's apparent affinity for InsP(3) m ainly by altering the rate constant for [H-3]InsP(3), dissociation. AT P (but not InsP(3)) also accelerated [H-3]InsP(3) dissociation. In con trast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [(3H)]LnsP(3).