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 SO²⁻_4i/Cl⁻_o exchange. Jennings (1995) also observed a ratio of SO²⁻₄ 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 ³⁵SO²⁻₄ 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 ³⁵SO²⁻₄ efflux). Experiments to address this issue are underway.