Rapid functional assays of recombinant IP3 receptors.
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).
Laude
Alex J
AJ
Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, UK.
Tovey
Stephen C
SC
Dedos
Skarlatos G
SG
Potter
Barry V L
BV
Lummis
Sarah C R
SC
Taylor
Colin W
CW
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000302:isolation & purification / Q000378:metabolism*; D020013:Calcium Signaling; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D015220:Calcium Channels; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium