Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP(3)) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP(3) receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace H-3-InsP(3) from its receptor and that they differ substantially in their affinities for the InsP(3) receptor (IC50 = 6.5-137 mu M). Recombinant type 1 InsP(3) receptors expressed in Sf9 cells were used to examine H-3-InsP(3) binding in cytosol-like medium: both fura-2 (IC50 = 796+/-86 mu M) and Ca Green-5N (IC50 = 62+/-7 mu M) completely inhibited the binding, but only in their Ca2+-free forms. Similar results were obtained with type 3 InsP(3) receptors. We conclude that many Ca2+ indicators in their Ca2+-free forms compete with InsP(3) for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.