Quantal responses to inositol 1,4,5-trisphosphate are not a consequence of Ca2+ regulation of inositol 1,4,5-trisphosphate receptors

Abstract

Submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) rapidly release only a fraction of the InsP3-sensitive intracellular Ca2+ stores, despite the ability of further increases in InsP3 concentration to evoke further Ca2+ release. The mechanisms underlying such quantal Ca2+ mobilization are not understood, but have been proposed to involve regulatory effects of cytosolic Ca2+ on InsP3 receptors. By examining complete concentration-effect relationships for InsP3-stimulated 45Ca2+ efflux from the intracellular stores of permeabilized hepatocytes, we demonstrate that, at 37 degrees C, responses to InsP3 are quantal in Ca(2+)-free media heavily buffered with either EGTA or BAPTA [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid]. Lower concentrations of InsP3 were used to examine the kinetics of Ca2+ mobilization at 2 degrees C, because at the lower temperature the stores were more sensitive to InsP3: the concentration of InsP3 causing half-maximal Ca2+ release (EC50) after a 30 s incubation decreased from 281 +/- 37 nM at 37 degrees C to 68 +/- 3 nM at 2 degrees C. At 2 degrees C, the EC50 for InsP3-stimulated Ca2+ mobilization decreased as the duration of exposure to InsP3 was increased: the EC50 was 68 +/- 3 nM after 30 s, and 29 +/- 2 nM after 420 s. InsP3-stimulated Ca2+ mobilization is therefore non-quantal at 2 degrees C: InsP3 concentration determines the rate, but not the extent, of Ca2+ release. By initiating quantal responses to InsP3 at 37 degrees C and then simultaneously diluting and chilling cells to 2 degrees C, we demonstrated that the changes that underlie quantal responses do not rapidly reverse at 2 degrees C. At both 37 degrees C and 2 degrees C, modest increases in cytosolic Ca2+ increased the sensitivity of the stores to InsP3, whereas further increases were inhibitory; both Ca2+ effects persisted after prior removal of ATP. We conclude that the effects of Ca2+ on InsP3 receptors are unlikely either to be enzyme-mediated or to underlie the quantal pattern of Ca2+ release evoked by InsP3.