Transfection of an inducible trout anion exchanger (AE1) into HEK-EcR cells.
A permanent cell line with inducible expression of the trout anion exchanger protein (trAE1) was constructed in a derivative of human embryonic kidney cells (HEK-293). In the absence of the inducer, muristerone A, the new cell line had no detectable trAE1 protein by Western analysis, biotinylation, and (36)Cl(-) flux. The amount of trAE1 protein increased with increasing dose and incubation time with muristerone A. Anion exchange inhibitors significantly inhibited the inducible flux of anions (i.e., (36)chloride and (35)sulfate) and taurine in isotonic media. The transfected cells had the characteristics of trAE1-mediated transport in intact trout erythrocytes: (1) inhibition by anion transport inhibitors, (2) pH independence over the pH range of 6.5-7.5, and (3) activation of (35)sulfate efflux by external anions in the selective order of Cl > Br > I > or = F. These cells, in contrast to trout erythrocytes, were not sensitive to the anion exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), suggesting some difference in the properties of the transfected AE1. These results demonstrate the inducible expression of new anion transport membrane protein in HEK-293 cells. This is the first expression of trAE1 in a mammalian system.
Davis
Erin M
EM
Brown University, Department of Molecular Pharmacology, Physiology and Biotechnology, Providence, Rhode Island 02912, USA.
Musch
mark W
mW
Goldstein
Leon
L
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D019921:Biotinylation; D015153:Blotting, Western; D002478:Cells, Cultured; D002713:Chlorine / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects; D007668:Kidney / Q000166:cytology; D010957:Plasmids / Q000235:genetics; D013431:Sulfates / Q000378:metabolism; D013654:Taurine / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014337:Trout / Q000235:genetics*
D017920:Antiporters; D013431:Sulfates; D013654:Taurine; D002713:Chlorine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid