Functional expression of the rat anion exchanger AE2 in insect cells by a recombinant baculovirus.
We used baculovirus to transiently express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. No detectable Cl(-)-HCO3- exchange activity was observed in wild type or sham-infected Sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a pH-sensitive fluorescent dye. Functional expression of anion exchange activity in the AE2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. The expressed AE2 anion exchange activity was Na+ independent and could be reversibly and irreversibly inhibited by the specific anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The reversible inhibition was sensitive to the concentration of DIDS, with a half inhibition of 4 microM. These results indicate that the rat AE2 protein produced in the recombinant baculovirus-infected insect cells is inserted into the plasma membrane in a biologically active form that appears suitable for functional studies of AE2.
He
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Department of Biochemistry, University of Rochester, New York 14642.
Wu
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Knauf
P A
PA
Tabak
L A
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Melvin
J E
JE
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins; D017920:Antiporters; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D007700:Kinetics; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D009036:Moths; D051381:Rats; D064606:SLC4A Proteins; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014162:Transfection
D027321:Anion Transport Proteins; D017920:Antiporters; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D064606:SLC4A Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D004247:DNA; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid