CA2-TRIPHOSPHATE BINDING IN MYELOID CELLS( AND MG2+ REGULATION OF INOSITOL 1,4,5)

Inhibition of inositol 1,4,5-triphosphate (Ins(1,4,5)P3) binding by Ca 2+ and Mg2+ remains a controversial issue: the Ca2+ effect may also be explained by Ins(1,4,5)P3 generation due to a Ca2+-sensitive phosphol ipase C, and the Mg2+ effect by Ins(1,4,5)P3 degradation. In this stud y, we have, therefore, investigated the effects of Ca2+ and Mg2+ on In s(1,4,5)P3 binding to membranes of the myeloid cell line HL-60 and ass ayed in parallel phospholipase C activity and Ins(1,4,5)P3 degradation . The assays for Ins(1,4,5)P3 binding, Ins(1,4,5)P3 generation, and In s(1,4,5)P3 degradation were performed under identical experimental con ditions. Ca2+ significantly and reversibly decreased Ins(1,4,5)P3 bind ing at submicromolar concentrations. Ca2+ stimulated Ins(1,4,5)P3 gene ration only at concentrations above 1 muM. At 1 muM [Ca2+], Ins(1,4,5) P3 binding was inhibited by 46 t 6%, but no Ins(1,4,5)P3 generation wa s observed. [Mg2+] between 0 and 1 mm slightly stimulated, while highe r concentrations inhibited, Ins(1,4,5)P3 binding. Mg2+ did not activat e phospholipase C. Neither Ca2+ nor Mg2+ induced relevant Ins(1,4,5)P3 degradation under the conditions of the binding assay. The effects of Ca2+ and Mg2+ on Ins(1,4,5)P3 binding were conserved after solubiliza tion of HL-60 membranes. However, only Mg2+, but not Ca2+, inhibited I ns(1,4,5)P3 binding to the receptor that had been partially purified b y heparin affinity chromatography. The Ca2+ sensitivity of Ins(1,4,5)P 3 binding to the partially purified receptor could be reconstituted by addition of the flow-through of the heparin column. No Ca2+-dependent Ins(1,4,5)P3 generation was observed in the reconstituted system. Thu s, we conclude that both Ca2+ and Mg2+ can inhibit Ins(1,4,5)P3 bindin g independently of phospholipase C activation and Ins(1,4,5)P3 breakdo wn. The Ca2+ effect depends upon factors that can be separated biochem ically from the Ins(1,4,5)P3 receptor. Mg2+ acts either directly on th e Ins(1,4,5)P3 receptor or on a closely associated target.