Stable overexpression of the type-1 inositol 1,4,5-trisphosphate receptor in L fibroblasts: subcellular distribution and functional consequences.
InsP3 receptor (InsP3R)/Ca(2+)-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 InsP3R 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.
Mackrill
J J
JJ
Department of Cell Physiology and Pharmacology, University of Leicester, U.K.
Wilcox
R A
RA
Miyawaki
A
A
Mikoshiba
K
K
Nahorski
S R
SR
Challiss
R A
RA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism; D007739:L Cells; D051379:Mice; D018160:Receptors, Cytoplasmic and Nuclear / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D015220:Calcium Channels; D018076:DNA, Complementary; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; D000252:Calcium-Transporting ATPases; D002118:Calcium