Reconstitution of the Na⁺/H⁺ Antiporter : A New Method for the Determination of H⁺ Efflux from Na⁺/H⁺ Antiporter-Reconstituted Vesicles

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A Na⁺/H⁺ antiporter was solubilized with octyl-β-D-glucopyranoside from rat renal brush border membrane and reconstituted in egg phosphatidylcholine liposomes. The activity of the antiporter was determined by a new method. Reconstitution was performed in a solution containing a pH-sensitive fluorescence dye, fluorescein isothiocyanatc-dextran (FITC-dextran), at pH 5.5. The reconstituted vesicles, encapsulating FITC-dextran, were incubated at pH 8.0, and the intravesicular pH (pH_i) was determined by monitoring fluorescence change of the dye. An increase in the fluorescence intensity which corresponded to an increase in pH_i was observed. The rate of the increase was dependent on the concentration of the extravesicular Na⁺, and the efflux of H⁺ in the presence of 150 mM sodium gluconate was suppressed by the addition of amiloride. These results show that the Na⁺/H⁺ antiporter was functionally reconstituted and that its activity can be determined simply by the use of FITC-dextran.