Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line.
A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.
Lee
B S
BS
Department of Biological Sciences, Stanford University, California 94305.
Gunn
R B
RB
Kopito
R R
RR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D027321:Anion Transport Proteins; D000838:Anions / Q000737:chemistry; D017920:Antiporters; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D064606:SLC4A Proteins; D014162:Transfection
D027321:Anion Transport Proteins; D000838:Anions; D017920:Antiporters; D008565:Membrane Proteins; D064606:SLC4A Proteins; C489237:SLC4A3 protein, human; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006859:Hydrogen; D004247:DNA; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid