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.
Electrogenic Sulfate/Chloride Exchange in Xenopus Oocytes Mediated by Murine AE1 E699Q
Address correspondence to Dr. S.L. Alper, Molecular Medicine Unit, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Fax: 617-667-2913; E-mail: [email protected]
Portions of this work were presented at the Forty-ninth annual meeting of the Society of General Physiologists (1995. J. Gen. Physiol. 106: 31a) and at the Twenty-eighth annual meeting of American Society of Nephrology (1995. J. Am. Soc. Nephrol. 6:303a).
Abbreviations used in this paper: AE1, anion exchanger 1; DNDS, 4,4′-dinitrostilbene-2,2′-disulfonic acid; WRK, Woodward's Reagent K (N-ethyl-5-phenylisoxazolium 3′-sulfonate); wt, wild-type.
Exclusion of the “outlier” experiment 2 from the calculations of the flux ratios resulted only in small changes. Such an edited mean of the individual flux ratios, 1.30 ± 0.19, did not differ statistically from the value in Table I. Such an edited ratio of the individual experimental mean flux values, 1.24 ± 0.05, differed slightly from that in Table I, but both values were consistent with a 1:1 stoichiometry of SO42−i/Cl−o exchange. Jennings (1995) also observed a ratio of SO42− efflux to Cl− influx of 1.21 ± 0.1 (SEM) in WRK-BH4–treated human red cells in the presence of gramicidin (Table 1 of Jennings, 1995).
Though AE1 E699Q-mediated inward current at clamped resting potential was of sufficient magnitude to conclude that all anion exchange flux measured under open circuit conditions was electrogenic (Table IV), extracellular isethionate supported a rate of AE1 E699Q-mediated 35SO42− efflux of 0.2 relative to extracellular Cl− (Fig. 5) but supported no greater inward current than did extracellular sulfate. Several factors might account for this discrepancy without invoking an alternate transport mechanism. First, current values of 0.2 relative to those recorded in extracellular Cl− are near or below the threshold of detection against the variable background current of individual oocytes. Second, the two assays were performed under different experimental conditions. Sulfate efflux into extracellular isethionate (Fig. 5) was measured 10 min after injection of oocytes with 50 nl carrier-free Na235SO4 in 50 mM Na HEPES, pH 7.4, whereas membrane potential change in response to extracellular anion substitution (Fig. 8, Table III) was measured in oocytes maintained in isethionate medium for 1–6 h after injection with 50 nl of 130 mM Na2SO4, 50 mM Na HEPES, pH 7.4. Lastly, evaluation of the effect of extracellular isethionate on the membrane potential of AE1 E699Q-expressing oocytes requires comparison with the effect of an extracellular monovalent anion to which the oocyte is “absolutely” or maximally impermeable (at least as judged by 35SO42− efflux). Experiments to address this issue are underway.
M.N. Chernova, L. Jiang, M. Crest, M. Hand, D.H. Vandorpe, K. Strange, S.L. Alper; Electrogenic Sulfate/Chloride Exchange in Xenopus Oocytes Mediated by Murine AE1 E699Q . J Gen Physiol 1 March 1997; 109 (3): 345–360. doi: https://doi.org/10.1085/jgp.109.3.345
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