INFLUENCE OF MG-2+ AND PH ON NMR-SPECTRA AND RADIOLIGAND BINDING OF INOSITOL 1,4,5-TRISPHOSPHATE

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 H-1- and P-31-n.m.r. spectroscopy to study whether these effects result 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 pK(a) of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pK(a) values for the 4-phosphate determined by P-31-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. H-1- and P-31-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-mu-M, 572-mu-M 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.