A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol polyphosphates

Abstract

This protocol describes a method for efficient chemical synthesis of an analog of inositol-1,4,5-trisphosphate (IP₃) hexakis acetoxymethyl ester having an ortho-nitroveratryl photochemical caging group on the 6-hydroxyl position. The six esters render the probe membrane permeant, such that it can be loaded into intact living cells in vitro or in vivo. Inside cells, the caged IP₃ is inert until activated by two-photon excitation at 720 nm. The photoliberated signaling molecule can mobilize release of Ca²⁺ from intracellular stores on the endoplasmic reticulum. When co-loaded with the fluorescent Ca²⁺ indicator rhod-2, one laser can be used for stimulating and monitoring intracellular Ca²⁺ signaling with single-cell resolution. This protocol has chemistry and biology sections; the former describes the organic synthesis of the caged IP₃, which requires 12 d, and the latter an application to a day-long study of astrocyte-regulated neuronal function in living brain slices acutely isolated from rats. As Ca²⁺ is the single most important intracellular second messenger and the IP₃-Ca²⁺ signaling cascade is used by many cells to produce increases in Ca²⁺ concentration, this method should be widely applicable for the study of a variety of physiological processes in intact biological systems.