Richardson A, Taylor C W
Department of Pharmacology, University of Cambridge, United Kingdom.
J Biol Chem. 1993 Jun 5;268(16):11528-33.
Changes in medium free [Ca2+] ([Ca2+]m) (< or = 1 microM) did not affect binding of [3H]InsP3 to inositol 1,4,5-trisphosphate (InsP3) receptors purified from cerebellum, but the chelators normally used to buffer [Ca2+]m inhibited InsP3 binding. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (1 mM) fully and reversibly inhibited specific [3H]InsP3 (0.6 nM) binding; the half-maximal effect (IC50) occurred with 0.34 +/- 0.03 mM BAPTA. Similar effects were observed with fura-2 (IC50 = 0.12 +/- 0.03 mM) and EDTA (IC50 = 8.7 +/- 1.9 mM). The order of potency of these Ca2+ chelators in inhibiting InsP3 binding was the reverse of their relative affinities for Ca2+; Ca2+ chelation is not, therefore, the cause of the inhibition. When [Ca2+]m was fixed (250-280 nM), InsP3-stimulated 45Ca2+ mobilization from permeabilized hepatocytes was competitively inhibited by increasing concentrations of BAPTA (KD = 1.8 mM). The effects of BAPTA were not a consequence of chelation of polyvalent cations, because TPEN (0.1 mM), which chelates heavy metal ions, did not affect InsP3 binding and pretreatment of all media with a polymetal sponge (DTPA-polyacrylamide) did not affect the sensitivity of InsP3 binding to BAPTA. We conclude that BAPTA and related Ca2+ chelators, in their Ca2+ -free forms, are competitive antagonists of InsP3 binding to its receptor.
培养基中游离钙离子浓度（[Ca2+]m）（≤1微摩尔）的变化并不影响从大脑小脑中纯化得到的三磷酸肌醇（InsP3）受体与[3H]InsP3的结合，但通常用于缓冲[Ca2+]m的螯合剂会抑制InsP3的结合。1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）（1毫摩尔）能完全且可逆地抑制特异性[3H]InsP3（0.6纳摩尔）的结合；半数最大效应（IC50）出现在0.34±0.03毫摩尔的BAPTA浓度时。用fura-2（IC50 = 0.12±0.03毫摩尔）和乙二胺四乙酸（EDTA）（IC50 = 8.7±1.9毫摩尔）时观察到类似的效应。这些钙离子螯合剂抑制InsP3结合的效力顺序与其对钙离子的相对亲和力顺序相反；因此，钙离子螯合不是抑制的原因。当[Ca2+]m固定在250 - 280纳摩尔时，InsP3刺激通透化肝细胞释放45Ca2+的过程会被浓度不断增加的BAPTA竞争性抑制（解离常数KD = 1.8毫摩尔）。BAPTA的作用并非多价阳离子螯合的结果，因为螯合重金属离子的三（2-吡啶甲基）胺（TPEN）（0.1毫摩尔）并不影响InsP3的结合，并且用多金属海绵（二乙烯三胺五乙酸 - 聚丙烯酰胺）对所有培养基进行预处理也不影响InsP3结合对BAPTA的敏感性。我们得出结论，BAPTA及相关的钙离子螯合剂在其无钙形式下是InsP3与其受体结合的竞争性拮抗剂。
