|
pubmed:abstractText |
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.
|
