Rapid functional assays of intracellular Ca2+ channels.
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.
Tovey
Stephen C
SC
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
Sun
Yi
Y
Taylor
Colin W
CW
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D007425:Intracellular Membranes / Q000502:physiology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors