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(+)/SO(4) (2−) 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 SO(4) (2−). E699Q-mediated Cl(−) influx was activated by elevation of intracellular SO(4) (2−), but E699Q-mediated Cl(−) efflux was undetectable. The DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid) sensitivity of E699Q-mediated SO(4) (2−) efflux was indistinguishable from that of wt AE1-mediated Cl(−) efflux. The extracellular anion selectivity of E699Q-mediated SO(4) (2−) efflux was similar to that of wt AE1-mediated Cl(−) efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl(−) with intracellular SO(4) (2−) was 1:1. Whereas SO(4) (2−) injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO(4) (2−), 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 SO(4) (2−)-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl(−)-dependent depolarization of SO(4) (2−)-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 (35)SO(4) (2−) efflux. Thus, AE1 E699Q-mediated SO(4) (2−)/ 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 SO(4) (2−)/Cl(−) exchange stoichiometry.