Rapid functional assays of intracellular Ca2+ channels

• DOI: 10.1038/nprot.2006.40
• Publication Date: 2006-07-27T20:11:12Z
• Authors: Stephen C Tovey; Yi Sun; Colin W Taylor
• Keywords: 0301 basic medicine; 03 medical and health sciences; Spectrometry, Fluorescence; Animals; Inositol 1,4,5-Trisphosphate Receptors; Calcium Channels; Intracellular Membranes; Endoplasmic Reticulum; Chickens; Cell Line

Functional assays of intracellular Ca2+ channels, such as the inositol 1,4,5-trisphosphate receptor (IP3R), have generally used 45Ca2+-flux assays, fluorescent indicators loaded within either the cytosol or the endoplasmic reticulum (ER) of single cells, or electrophysiological analyses. None of these methods is readily applicable to rapid, high-throughput quantitative analyses. Here we provide a detailed protocol for high-throughput functional analysis of native and recombinant IP3Rs. A low-affinity Ca2+ indicator (mag-fluo-4) trapped within the ER of permeabilized cells is shown to report changes in luminal free Ca2+ concentration reliably. An automated fluorescence plate reader allows rapid measurement of Ca2+ release from intracellular stores mediated by IP3R. The method can be readily adapted to other cell types or to the analysis of other intracellular Ca2+ channels. This protocol can be completed in 2-3 h.