Rapid filtration studies of the effect of cytosolic Ca2+ on inositol 1,4,5-trisphosphate-induced 45Ca2+ release from cerebellar microsomes

Abstract

Using microsomal membrane vesicles derived from sheep cerebellum, we measured the rate of inositol 1,4,5-trisphosphate (InsP3)-dependent 45Ca2+ efflux from 45Ca(2+)-loaded compartments during rapid perfusion with a medium containing InsP3 and various concentrations of free 40Ca2+ on the cytosolic side (pH 7.1, 5 mM Mg2+, in the absence of ATP at 20 degrees C). At 0.15 microM InsP3 and pCa 6.5, half-45Ca2+ release was attained within less than 200 ms. At low Ca2+ concentrations, the initial rate of 45Ca2+ release depended smoothly on InsP3 concentration, and InsP3 activated release with moderate positive cooperativity. Preliminary experiments performed at various free 40Ca2+ concentrations were consistent with a bell-shaped 40Ca2+ dependence of 45Ca2+ release. In the range of micromolar or higher free 40Ca2+ concentrations, the apparent inhibition of 45Ca2+ release was dependent on InsP3 concentration, and 45Ca2+ release for intermediate InsP3 concentrations was transient; under selected conditions, a second perfusion period, identical to the first one but separated from it by a short recovery period, was found to allow renewed 45Ca2+ efflux. At high Ca2+ concentration, fast reversible Ca(2+)-dependent desensitization of the channel, and not heterogeneity, was therefore responsible for the termination of InsP3-triggered 45Ca2+ efflux at submaximal concentrations of InsP3. At lower Ca2+ concentrations, a large fraction of the apparent activating effect of submicromolar 40Ca2+ concentrations on 45Ca2+ efflux that we had observed in the preliminary experiments proved to be the artifactual consequence of an inhibitory effect exerted by metal-free Ca2+ chelators on InsP3-dependent efflux at nanomolar 40Ca2+ concentrations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3 were all effective inhibitors. When this inhibition was taken into account, a rise in free 40Ca2+ concentration from 30 to 300 nM only weakly enhanced 45Ca2+ fluxes in the presence of a low concentration of InsP3. As a result, submicromolar free 40Ca2+ appears to be only a poor activator of InsP3-induced Ca2+ release under these experimental conditions.