RECONSTITUTION OF THE Na^+/H^+ ANTIPORTER IN LIPOSOMES

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Na^+/H^+ antiporter is present on the plasma membrane of a wide variety of cells, and play an important role in regulating intracellular pH. The Na^+/H^+ antiporter is known to be activated by various stimuli such as growth factors and to cause intracellular alkalinization. Thus the purification and characterization of this antiporter are of interest for elucidation of signal transduction. In this study, Na^+/H^+ antiporter was solubilized with octyl-beta-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 isothiocyanate-dextran(FITC-dextran), at pH 5.5. The reconstituted vesicles encapsulating FITC-dextran were incubated at pH 8.0, and the intravesicular pH (pHi) was determined by monitoring fluorescence change of the dye. An increase in the fluorescence intensity which corresponded to an increase in pHi was observed. The rate of the increase was dependent on the concentration of the extravesicular Ne+, 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.In order to understand the mechanism of anesthesia, we also investigated the effect of local anesthetics on Na^+/H^+antiporter reconstituted in liposomes. Dibucaine and tetracaine decreased H^+ efflux irresrective of the presence of Na^+ gradient. On the other hand, lidocaine at 10 muM inhibited H^+ efflux in the presence of inward Na^+ gradient, indicating that the direct interaction of local anesthetics with Na^+/H^+ antiporter and/or other Na^+ transporters may be a trigger of anesthesia.