Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Inositol trisphosphate (InsP3) receptors are crucial for intracellular calcium signaling.
• Fluorescent calcium indicators like Fura-2 and BAPTA were previously noted as potential InsP3 receptor antagonists.
• Previous studies were limited by performing analyses at pH 8.3.

Purpose of the Study:

• To investigate the interaction between commonly used fluorescent Ca2+ indicators and InsP3 receptors at a physiological pH (7.2).
• To quantify the affinity of Ca2+ indicators for InsP3 receptors using a scintillation proximity assay (SPA).
• To determine if the Ca2+-bound or Ca2+-free form of indicators mediates the interaction.

Main Methods:

• Utilized a scintillation proximity assay (SPA) with pure cerebellar InsP3 receptors.
• Examined the binding of 3H-InsP3 to recombinant type 1 and type 3 InsP3 receptors expressed in Sf9 cells.
• Tested the effects of Ca2+-free and Ca2+-bound forms of fluorescent Ca2+ indicators (e.g., Fura-2, Ca Green-5N) on 3H-InsP3 binding.

Main Results:

• At pH 7.2, several fluorescent Ca2+ indicators reversibly displaced 3H-InsP3 from InsP3 receptors.
• The affinities of these indicators for the InsP3 receptor varied significantly (IC50 = 6.5–137 microM).
• Fura-2 and Ca Green-5N (in their Ca2+-free forms) inhibited 3H-InsP3 binding to both type 1 and type 3 InsP3 receptors, with Ca Green-5N showing higher affinity (IC50 = 62 microM).

Conclusions:

• Many Ca2+ indicators, particularly in their Ca2+-free state, act as competitive antagonists of InsP3 receptors.
• The interaction between Ca2+ indicators and InsP3 receptors occurs with sufficient affinity to potentially alter physiological responses.
• Researchers should be aware of this potential interference when using Ca2+ indicators in studies involving InsP3 receptor signaling.