High-Throughput Functional Assays of IP₃-Evoked Ca²⁺ Release

Abstract

This protocol describes procedures for high-throughput functional analyses of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in permeabilized cells. The methods are applicable to native IP₃Rs in a variety of cells and to recombinant IP₃Rs stably expressed in DT40 cells in which gene disruption has abolished expression of endogenous IP₃Rs. A low-affinity Ca²⁺-indicator (Mag-Fluo-4) trapped within the endoplasmic reticulum (ER) of permeabilized cells is used to report changes in luminal free Ca²⁺ concentration. A fluorescence plate-reader equipped to allow automated additions permits rapid measurements of the Ca²⁺ release evoked by IP₃R. The procedure can be completed in 2–3 h.