Advances in methods for analyzing IP₃ signaling and understanding of coupled Ca²⁺ and IP₃ oscillations

Abstract

Inositol 1,4,5-trisphosphate (IP₃) is an important intracellular messenger produced by phospholipase C via the activation of G-protein-coupled receptor- or receptor-tyrosine-kinase-mediated pathways, and is involved in numerous responses to hormones, neurotransmitters, and growth factors through the releases of Ca²⁺ from intracellular stores via IP₃ receptors. IP₃-mediated Ca²⁺ signals often exhibit complex spatial and temporal organizations, such as Ca²⁺ oscillations. Recently, new methods have become available to measure IP₃ concentration ([IP₃]) using AlphaScreen technology, fluorescence polarization, and competitive ligand binding assay (CFLA). These methods are useful for the high throughput screening in drug discovery. Calcium ions generate versatile intracellular signals such as Ca²⁺ oscillations and waves. Fluorescent sensors molecules to monitor changes in [IP₃] in single living cells are crucial to study the mechanism for the spatially and temporally regulated Ca²⁺ signals. In particular, FRET-based IP₃ sensors are useful for the quantitative monitoring intracellular [IP₃], and allowed to uncovered the oscillatory IP₃ dynamics in association with Ca²⁺ oscillations. A mathematical model of coupled Ca²⁺ and IP₃ oscillations predicts that Ca²⁺ oscillations are the result of modulation of the IP₃ receptor by intracellular Ca²⁺, and that the period is modulated by the accompanying IP₃ oscillations. These model predictions have also been confirmed experimentally. At present, however, usefulness of FRET-based IP₃ sensors are limited by their relatively small change in fluorescence. Development of novel IP₃ sensors with improve dynamic range would be important for understanding the regulatory mechanism of Ca²⁺ signaling and for in vivo IP₃ imaging.