Rapid functional assays of recombinant IP3 receptors

  • Laude A
  • Tovey S
  • Dedos S
 et al. 
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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 ??l). ?? 2005 Elsevier Ltd. All rights reserved.

Author-supplied keywords

  • Ca2+ indicator
  • Ca2+ mobilization
  • DT40 cell
  • IP3
  • Luminal Ca2+
  • Screening

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Authors

  • Alex J. Laude

  • Stephen C. Tovey

  • Skarlatos G. Dedos

  • Barry V L Potter

  • Sarah C R Lummis

  • Colin W. Taylor

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