Wilcox R A, Challiss R A, Baudin G, Vasella A, Potter B V, Nahorski S R
Department of Pharmacology and Therapeutics, University of Leicester, U.K.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):191-4. doi: 10.1042/bj2940191.
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.
在皂素通透处理的SH-SY5Y人神经母细胞瘤细胞中，1,3,4,5-肌醇四磷酸（Ins(1,3,4,5)P4）能够以浓度依赖的方式动员整个对1,4,5-肌醇三磷酸（Ins(1,4,5)P3）敏感的细胞内钙离子储存库，其半数有效浓度（EC50）值为2.05±0.45微摩尔，而Ins(1,4,5)P3的EC50值为0.14±0.03微摩尔。然而，L-1,3,4,5-肌醇四磷酸[=D-1,3,5,6-肌醇四磷酸]在浓度高达100微摩尔时未能引起细胞内钙离子的动员。以猪小脑膜作为Ins(1,4,5)P3/Ins(1,3,4,5)P4特异性结合位点的来源进行的结合研究揭示了它们在立体特异性要求上的显著差异。猪小脑的Ins(1,4,5)P3受体表现出严格的立体特异性，L-Ins(1,4,5)P3和L-Ins(1,3,4,5)P4的亲和力比D-Ins(1,4,5)P3弱1000倍以上；而在从一个明显均一的Ins(1,4,5)P3受体群体中取代特异性[3H]Ins(1,4,5)P3结合方面，Ins(1,3,4,5)P4（IC50为762±15纳摩尔）仅比D-Ins(1,4,5)P3（IC50为20.7±9.7纳摩尔）弱约40倍。相比之下，Ins(1,3,4,5)P4结合位点表现出较差的立体选择性。Ins(1,3,
