EFFECTS OF CA2-TRISPHOSPHATE (INSP3) RECEPTORS AND INSP3-STIMULATED CA2+ MOBILIZATION( CHELATORS ON PURIFIED INOSITOL 1,4,5)

Changes in medium free [Ca2+] ([Ca2+]m) (less-than-or-equal-to 1 muM) did not affect binding of [H-3]InsP3 to inositol 1,4,5-trisphosphate ( InsP3) receptors purified from cerebellum, but the chelators normally used to buffer [Ca2+]m inhibited InsP3 binding. ,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) (1 mM) fully and reversibly inhibited specific [H-3]InsP3 (0.6 nM) binding; the half-maximal effec t (IC50) occurred with 0.34 +/- 0.03 mM BAPTA. Similar effects were ob served 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 In sP3 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 Ca-45(2+) mobilization fro m permeabilized hepatocytes was competitively inhibited by increasing concentrations of BAPTA (K(D) = 1.8 mM). The effects of BAPTA were not a consequence of chelation of polyvalent cations, because TPEN (0.1 m M), 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 conclud e that BAPTA and related Ca2+ chelators, in their Ca2+-free forms, are competitive antagonists of InsP3 binding to its receptor.