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Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).

Original publication

DOI

10.1016/j.ceca.2005.04.001

Type

Journal article

Journal

Cell Calcium

Publication Date

07/2005

Volume

38

Pages

45 - 51

Keywords

Animals, B-Lymphocytes, Calcium, Calcium Channels, Calcium Signaling, Chickens, Cytosol, Endoplasmic Reticulum, Fluorescent Dyes, Inositol 1,4,5-Trisphosphate, Inositol 1,4,5-Trisphosphate Receptors, Receptors, Cytoplasmic and Nuclear, Recombinant Proteins