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) 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 μM, compared with 0.14 ± 0.03 μM for Ins(1,4,5)Pa. 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 μM. 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(IC₅₀762 ± 15 nM) was only about 40-fold weaker than D-Ins(1,4,5)P₃ (IC₅₀ 20.7 ± 9.7nM) 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 K(D) values for high- and low-affinity sites of 2.1 ± 0.5 nM and 918 ± 161 nM respectively. L-Ins(1,3,4,5)P,L 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 Ca²⁺-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.