Electrogenic sulfate/chloride exchange in Xenopus oocytes mediated by murine AE1 E699Q

Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO42- cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl- influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO42-. E699Q-mediated Cl- influx was activated by elevation of intracellular SO42-, but E699Q-mediated Cl- efflux was undetectable. The DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) sensitivity of E699Q-mediated SO42- efflux was indistinguishable from that of wt AE1-mediated Cl- efflux. The extracellular anion selectivity of E699Q-mediated SO42- efflux was similar to that of wt AE1-mediated Cl- efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl- with intracellular SO42- was 1:1. Whereas SO42- injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO42-, but not of Cl- or gluconate, into oocytes expressing E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl- with isethionate caused a 28-mV hyperpolarization in SO42--loaded oocytes expressing E699Q but had no effect on oocytes expressing wt AE1. Extracellular Cl--dependent depolarization of SO42--preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl- was of magnitude sufficient to account for measured (SO42-)-S-35 efflux. Thus, AE1 E699Q-mediated SO42-/Cl- exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO42-/Cl- exchange stoichiometry.