Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes.
cRNA encoding the murine band 3-related protein AE2 was expressed in Xenopus oocytes. AE2-mediated transport function and regulation were analyzed by unidirectional 36Cl- influx and efflux studies. AE2 cRNA-injected oocytes took up 36Cl- as much as 40-fold faster than did water-injected oocytes. AE2-mediated 36Cl- uptake increased as a function of increasing uptake time, number of days after cRNA injection, and amount of injected cRNA. Among the functional properties of AE2 evaluated were transport mechanism and substrate specificity, inhibitor pharmacology, and regulation by pH. The apparent Km for external Cl- was 5.6 mM. AE2 was defined as a Cl-/anion exchanger by two criteria: 1) 36Cl- efflux from AE2-expressing oocytes was maximally stimulated by extracellular Cl- or nitrate; AE2-associated 36Cl- efflux was supported by substitution of extracellular Cl- with other anions in the rank order bromide > isethionate > or = gluconate > iodide and 2) prolonged preincubation of AE2 cRNA-injected oocytes in Cl(-)-free media containing isethionate, gluconate, or glutamate decreased subsequent AE2-associated 36Cl- uptake from Cl- media in rough proportion to the degree of intracellular Cl- depletion, whereas preincubation in nitrate medium had no effect. AE2-associated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid at half-maximally inhibitory concentrations between 0.5 and 19 microM, depending on extracellular Cl- concentration, and progressed to irreversibility at 20 degrees C with a half-time of 20-30 min. Many additional inhibitors showed lower potency for AE2 than previously reported for AE1. Although AE2 expression did not change oocyte resting intracellular pH, AE2-associated 36Cl- influx and efflux were each decreased in acid incubation medium and increased in alkaline medium.
Molecular Medicine Unit, Beth Israel Hospital, Boston, Massachusetts.
B D
BD
Humphreys
L
L
Jiang
M N
MN
Chernova
S L
SL
Alper
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions / Q000378:metabolism; D017920:Antiporters; D002712:Chlorides / Q000378:metabolism; D003470:Culture Media; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007474:Ion Exchange; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009865:Oocytes / Q000378:metabolism*; D064606:SLC4A Proteins; D014981:Xenopus
D027321:Anion Transport Proteins; D000838:Anions; D017920:Antiporters; D002712:Chlorides; D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D064606:SLC4A Proteins; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid