Rapid functional assays of recombinant IP3 receptors

Abstract

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 IP3R, and establish that the indicator is capable of reliably reporting the Ca2+ 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 (≤100 μl). © 2005 Elsevier Ltd. All rights reserved.