Incremental Ca2+ mobilization by inositol trisphosphate receptors is unlikely to be mediated by their desensitization or regulation by luminal or cytosolic Ca2+

The kinetics of Ins(1,4,5)P-3 (InsP(3))-stimulated Ca2+ release from intracellular stores are unusual in that submaximal concentrations of InsP(3) rapidly release only a fraction of the InsP(3)-sensitive Ca2+ stores. By measuring unidirectional Ca-45(2+) efflux from permeabilized rat hepatocytes, we demonstrate that such quantal responses to InsP(3) occur at all temperatures between 2 and 37 degrees C, but at much lower rates at the lower temperatures. Preincubation with submaximal concentrations of InsP(3), which themselves evoked quantal Ca2+ release, had no effect on the sensitivity of the stores to further additions of InsP(3). The final Ca2+ content of the stores was the same whether they were stimulated with two submaximal doses of InsP(3) or a single addition of the sum of these doses. Such incremental responses and the persistence of quantal behaviour at 2 degrees C indicate that InsP(3)-evoked receptor inactivation is unlikely to be the cause of quantal Ca2+ mobilization. Reducing the Ca2+ content of the intracellular stores by up to 45% did not affect their sensitivity to InsP(3), but substantially reduced the time taken for each submaximal InsP(3) concentration to exert its full effect. These results suggest that neither luminal nor cytosolic Ca2+ regulation of InsP(3) receptors are the determinants of quantal behaviour. Our results are not therefore consistent with incremental responses to InsP(3) depending on mechanisms involving attenuation of InsP(3) receptor function by cytosolic or luminal Ca2+ or by InsP(3) binding itself. We conclude that incremental activation of Ca2+ release results from all-or-nothing emptying of stores with heterogeneous sensitivities to InsP(3). These characteristics allow rapid graded recruitment of InsP(3)-sensitive Ca2+ stores as the cytosolic InsP(3) concentration increases.