Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction.
The relative expression of different inositol 1,4,5-trisphosphate receptor (InsP3R) mRNA was determined in a selection of murine and rat cell types that are commonly used to study InsP3-mediated Ca2+ signaling. Different mRNA species (encoding the known InsP3R isoforms) were co-amplified using common polymerase chain reaction primer pairs that recognized sequences that are totally conserved between the various InsP3R. Specific identification of the co-amplified sequences was done by restriction site analysis. In cerebellum, mRNA encoding InsP3R-I accounted for > 90% of the total InsP3R mRNA. This isoform was also present in all other cell types tested and was often the major isoform. In contrast, the level of expression of the other isoforms was cell type-specific. A new InsP3R isoform (type V) was detected that had 94.5% sequence identity with the InsP3R-II in the amplified region. Interestingly, this isoform was largely expressed in murine but not in rat cells. We functionally characterized InsP3R-V using the mouse fibroblast C3H10T1/2 cells, where mRNA encoding InsP3R-V accounted for 76.4% of the total InsP3R mRNA. InsP3-induced Ca2+ release in permeabilized C3H10T1/2 cells was regulated by luminal and cytosolic Ca2+, stimulated by thimerosal, and inhibited by caffeine.
De Smedt
H
H
Laboratory of Physiology, Campus Gasthuisberg O/N, K.U.Leuven, Belgium.
Missiaen
L
L
Parys
J B
JB
Bootman
M D
MD
Mertens
L
L
Van Den Bosch
L
L
Casteels
R
R
D000818:Animals; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics* / Q000302:isolation & purification; D002478:Cells, Cultured; D002531:Cerebellum / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D014644:Genetic Variation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000302:isolation & purification; D015398:Signal Transduction; D013849:Thimerosal / Q000494:pharmacology; D014018:Tissue Distribution
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D013849:Thimerosal; D002110:Caffeine; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium