White A M, Varney M A, Watson S P, Rigby S, Liu C S, Ward J G, Reese C B, Graham H C, Williams R J
Department of Pharmacology, University of Oxford, UK.
Biochem J. 1991 Sep 15;278 ( Pt 3)(Pt 3):759-64. doi: 10.1042/bj2780759.
We and others have shown that the binding of Ins(1,4,5)P3 to its receptor is pH-sensitive and can be inhibited by Mg2+. In the present study we have used 1H- and 31P-n.m.r. spectroscopy to study whether these effects results from increased ionization of Ins(1,4,5)P3 and a direct interaction with Mg2+ respectively. Under near-physiological conditions of ionic strength (100 mM-KCl), three ionizable groups were observed. The pH titration curve of the 1-phosphate was monophasic, with a pKa of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pKa values for the 4-phosphate determined by 31P-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. 1H- and 31P-n.m.r. measurements suggest that Mg2+ binds weakly to Ins(1,4,5)P3 at physiological pH. Mg2+ non-competitively inhibited binding of Ins(1,4,5)P3 to its receptor in rat cerebellum and bovine adrenal cortex. Inhibition curves for rat cerebellum at pH 7.1 and 8.5, and also for bovine adrenal cortex at pH 8.5, appeared to be monophasic, with IC50 values (concn. of displacer giving 50% inhibition of specific binding) of 214 microM, 572 microM and 9.1 mM respectively. Scatchard analysis revealed that Mg2+ inhibited binding of Ins(1,4,5)P3 to bovine adrenal cortex at pH 8.5 in a non-competitive manner. Our results suggest that the previously reported pH-sensitivity of the binding of Ins(1,4,5)P3 may be caused by ionization of the phosphate groups in positions 4 and 5, and that the ability of Mg2+ to inhibit the binding of Ins(1,4,5)P3 is not mediated by direct chelation but through a site located on, or close to, the Ins(1,4,5)P3 receptor. Inhibition by Mg2+ is pH-sensitive and can vary at least 10-fold between tissues, suggesting possible receptor heterogeneity. Mg2+ may exert an important regulatory control on the release of Ca2+ by Ins(1,4,5)P3.
我们及其他研究人员已表明，肌醇三磷酸（Ins(1,4,5)P3）与其受体的结合对pH敏感，且可被Mg2+抑制。在本研究中，我们利用1H-和31P-核磁共振光谱来研究这些效应是否分别源于Ins(1,4,5)P3离子化增加以及与Mg2+的直接相互作用。在接近生理离子强度（100 mM - KCl）的条件下，观察到三个可离子化基团。1-磷酸基团的pH滴定曲线呈单相，pKa为6.3。4-磷酸基团和5-磷酸基团的滴定曲线呈双相，表明这些基团相互作用；通过31P-核磁共振测定的4-磷酸基团的pKa值为5.7和7.8，5-磷酸基团的pKa值为5.3和7.9。1H-和31P-核磁共振测量表明，在生理pH条件下，Mg2+与Ins(1,4,5)P3的结合较弱。Mg2+以非竞争性方式抑制Ins(1,4,5)P3与大鼠小脑和牛肾上腺皮质中其受体的结合。在pH 7.1和8.5时大鼠小脑以及在pH 8.5时牛肾上腺皮质的抑制曲线似乎呈单相，其半数抑制浓度（IC50值，即导致特异性结合抑制50%的置换剂浓度）分别为214 microM、572 microM和9.1 mM。Scatchard分析表明，在pH 8.5时，Mg2+以非竞争性方式抑制Ins(1,4,5)P3与牛肾上腺皮质的结合。我们的结果表明，先前报道的Ins(1,4,5)P3结合的pH敏感性可能是由4位和5位磷酸基团的离子化引起的，并且Mg2+抑制Ins(1,4,5)P3结合的能力不是通过直接螯合介导的，而是通过位于Ins(1,4,5)P3受体上或其附近的一个位点介导的。Mg2+的抑制作用对pH敏感，且在不同组织之间至少可相差10倍，这表明可能存在受体异质性。Mg2+可能对Ins(1,4,5)P3介导的Ca2+释放发挥重要的调节控制作用。
