Stereoselectivity of Ins(1,3,4,5)P4 recognition sites: implications for the mechanism of the Ins(1,3,4,5)P4-induced Ca2+ mobilization.

Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in saponin-permeabilized SH-SY5Y human neuroblastoma cells in a concentration-dependent manner, yielding an EC50 value of 2.05 +/- 0.45 microM, compared with 0.14 +/- 0.03 microM for Ins(1,4,5)P3. However, L-Ins(1,3,4,5)P4 [= D-Ins(1,3,5,6)P4] failed to cause mobilization of intracellular Ca2+ at concentrations up to 100 microM. Binding studies using pig cerebellar membranes as a source of both Ins(1,4,5)P3/Ins(1,3,4,5)P4-specific binding sites have revealed a marked contrast in their stereospecificity requirements. Ins(1,4,5)P3-receptors from pig cerebella exhibited stringent stereospecificity, L-Ins(1,4,5)P3 and L-Ins(1,3,4,5)P4 were > 1000-fold weaker, whereas Ins(1,3,4,5)P4 (IC50 762 +/- 15 nM) was only about 40-fold weaker than D-Ins(1,4,5)P3 (IC50 20.7 +/- 9.7 nM) at displacing specific [3H]Ins(1,4,5)P3 binding from an apparently homogeneous Ins(1,4,5)P3 receptor population. In contrast, the Ins(1,3,4,5)P4-binding site exhibited poor stereoselectivity. Ins(1,3,4,5)P4 produced a biphasic displacement of specific [32P]Ins(1,3,4,5)P4 binding, with two-site analysis revealing KD values for high- and low-affinity sites of 2.1 +/- 0.5 nM and 918 +/- 161 nM respectively. L-Ins(1,3,4,5)P4 also produced a biphasic displacement of specific [32P]Ins(1,3,4,5)P4 binding which was less than 10-fold weaker than with D-Ins(1,3,4,5)P4 (IC50 values for the high- and low-affinity sites of 17.2 +/- 3.7 nM and 3010 +/- 542 nM respectively). Therefore, although L-Ins(1,3,4,5)P4 appears to be a high-affinity Ins(1,3,4,5)P4-binding-site ligand in pig cerebellum, it is a very weak agonist at the Ca(2+)-mobilizing receptors of permeabilized SH-SY5Y cells. We suggest that the ability of D-Ins(1,3,4,5)P4 to access intracellular Ca2+ stores may derive from specific interaction with the Ins(1,4,5)P3- and not the Ins(1,3,4,5)P4-receptor population.