STEREOSELECTIVITY OF INS(1,3,4,5)P(4) RECOGNITION SITES - IMPLICATIONS FOR THE MECHANISM OF THE INS(1,3,4,5)P(4)-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 neurob lastoma cells in a concentration-dependent manner, yielding an EC50 va lue of 2.05 +/- 0.45 muM, compared with 0.14 +/- 0.03 muM for Ins(1,4, 5)P3. However, L-Ins(1,3,4,5)P4 [= D-Ins(1,3,5,6)P4] failed to cause m obilization of intracellular Ca2+ at concentrations up to 100 muM. Bin ding 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 co ntrast 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 [H-3]Ins(1,4,5)P3 bi nding from an apparently homogeneous Ins(1,4,5)P3 receptor population. In contrast, the Ins(1,3,4,5)P4-binding site exhibited poor stereosel ectivity. Ins(1,3,4,5)P4 produced a biphasic displacement of specific [P-32]Ins(1,3,4,5)P4 binding, with two-site analysis revealing K(D)) v alues for high- and low-affinity sites of 2.1 +/- 0.5 nM and 918 +/- 1 61 nM respectively. L-Ins(1,3,4,5)P4 also produced a biphasic displace ment of specific [P-32]Ins(1,3,4,5)P4 binding which was less than 10-f old weaker than with D-Ins(1,3,4,5)P4 (IC50 values for the high- and l ow-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 I ns(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 cel ls. We suggest that the ability of D-Ins(1,3,4,5)P4 to access intracel lular 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.