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 cRNA. Among the functional properties of AE2 evaluated were transport mechanism and substrate specificity, inhibitor pharmacology, and regulation by pH. The apparent K(m) 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 > isothionate > gluconate > iodide and 2) prolonged preincubation of AE2 cRNA-injected oocytes in Cl^-- free media containing isothionate, 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 μM, depending on extracellular Cl^- concentration and progressed to irreversibility at 20°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.