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 Ca(2+)-indicator (Mag-Fluo-4) trapped within the endoplasmic reticulum (ER) of permeabilized cells is used to report changes in luminal free Ca(2+) concentration. A fluorescence plate-reader equipped to allow automated additions permits rapid measurements of the Ca(2+) release evoked by IP3R. The procedure can be completed in 2-3 h.