Electrogenic Sulfate/Chloride Exchange in Xenopus Oocytes Mediated by Murine AE1 E699Q

Abstract

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 ³⁵SO₄²⁻ 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.