STABLE OVEREXPRESSION OF THE TYPE-1 INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN L FIBROBLASTS - SUBCELLULAR-DISTRIBUTION AND FUNCTIONAL CONSEQUENCES

InsP(3) receptor (InsP(3)R)/Ca2+-release channels differ markedly in a bundance in different tissues/cell types and InsP(3)R expression level s may be modulated in response to a variety of external cues. Cell lin es overexpressing InsP(3)Rs will provide useful models for the study o f the influence of receptor density and subtype on InsP(3)-mediated Ca 2+ signalling We have investigated the properties of InsP(3)Rs in mous e L fibroblast cell lines transfected with either type-1 InsP(3)R cDNA (L15) or vector control (Lvec). L15 cells express approximately eight fold higher levels of the type-1 InsP(3)R protein than Lvec cells, as assessed by radioligand binding and immunoblotting. Increased expressi on was stable since it did not alter over ten cell passages. Both L15 and Lvec cells express predominantly the type-1 InsP(3)R isoform, indi cating that functional differences in the InsP(3)-mediated Ca2+ signal ling in these cell lines are due to alteration in the levels of recept or rather than changes in the isoform expressed. Type-1 InsP(3)R in L1 5 cells is largely associated with subcellular membrane fractions bear ing 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+ m obilization in response to increasing concentrations of Ins(1,4,5)P-3. This study indicates that L15/Lvec cells provide a suitable model for studying the effects of InsP(3)R expression level on InsP(3)-induced Ca2+ mobilization.