Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction

De Smedt, Humbert; Missiaen, Ludwig; Parys, Jan B.; Bootman, Martin D.; Mertens, Luc; Van Den Bosch, Ludo and Casteels, Rik (1994). Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. The Journal of Biological Chemistry, 269(34) pp. 21691–21698.

URL: http://www.jbc.org/content/269/34/21691.short

Abstract

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 C₃H10T½ cells, where mRNA encoding InsP₃R-V accounted for 76.4% of the total InsP₃R mRNA. InsP₃-induced Ca²⁺ release in permeabilized C₃H10T½ cells was regulated by luminal and cytosolic Ca²⁺, stimulated by thimerosal, and inhibited by caffeine.

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