Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes.

Abstract

Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.