Stable overexpression of the type-1 inositol 1,4,5-trisphosphate receptor in L fibroblasts: Subcellular distribution and functional consequences

InsP₃ receptor (InsP₃R)/Ca²⁺-release channels differ markedly in abundance in different tissues/cell types and InsP₃R expression levels may be modulated in response to a variety of external cues. Cell lines overexpressing InsP₃Rs will provide useful models for the study of the influence of receptor density and subtype on InsP₃-mediated Ca²⁺ signalling. We have investigated the properties of InsP₃Rs in mouse L fibroblast cell lines transfected with either type-1 InsP₃,R cDNA (L15) or vector control (Lvec). L15 cells express approximately eightfold higher levels of the type-1 InsP₃R protein than Lvec cells, as assessed by radioligand binding and immunoblotting. Increased expression was stable since it did not alter over ten cell passages. Both L15 and Lvec cells express predominantly the type-1 InsP₃R isoform, indicating that functional differences in the InsP₃-mediated Ca²⁺ signalling in these cell lines are due to alteration in the levels of receptor rather than changes in the isoform expressed. Type-1 InsP₃R in L15 cells is largely associated with subcellular membrane fractions bearing the sarco/endoplasmic reticulum Ca²⁺ ATPase pump, appropriate for rapidly exchanging Ca²⁺ pools. Functionally, there is an approximately fourfold increase in the sensitivity of permeabilized L15-cell Ca²⁺ mobilization in response to increasing concentrations of Ins(1,4,5)P₃. This study indicates that L15/Lvec cells provide a suitable model for studying the effects of InsP₃R expression level on InsP₃-induced Ca²⁺ mobilization.