Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators
| Autor: | Colin W. Taylor, Gerry O'Beirne, Vanessa Correa, Stephen A. Morris, Thomas J. A. Cardy |
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| Rok vydání: | 1999 |
| Předmět: |
Insecta
Physiology Recombinant Fusion Proteins Receptors Cytoplasmic and Nuclear Sf9 Biology law.invention chemistry.chemical_compound Cytosol BAPTA law Animals Inositol 1 4 5-Trisphosphate Receptors Receptor Molecular Biology Cells Cultured Fluorescent Dyes Dose-Response Relationship Drug Inositol trisphosphate Cell Biology Fluorescence Affinities Rats Kinetics Scintillation proximity assay Biochemistry chemistry Recombinant DNA Scintillation Counting Calcium Calcium Channels Baculoviridae Protein Binding |
| Zdroj: | Cell calcium. 25(2) |
| ISSN: | 0143-4160 |
| Popis: | Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (Ins P 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 Ins P 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 Ca 2+ indicators reversibly displace 3 H-Ins P 3 from its receptor and that they differ substantially in their affinities for the Ins P 3 receptor (IC 50 = 6.5–137 μM). Recombinant type 1 Ins P 3 receptors expressed in Sf9 cells were used to examine 3 H-Ins P 3 binding in cytosol-like medium: both fura-2 (IC 50 = 796±86 μM) and Ca Green-5N (IC 50 = 62±7 μM) completely inhibited the binding, but only in their Ca 2+ -free forms. Similar results were obtained with type 3 Ins P 3 receptors. We conclude that many Ca 2+ indicators in their Ca 2+ -free forms compete with Ins P 3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses. |
| Databáze: | OpenAIRE |
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