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

Na^+/H^+反转运蛋白存在于多种细胞的质膜上，在调节细胞内ph中起重要作用。Na^+/H^+反转运蛋白可被多种刺激（如生长因子）激活并引起细胞内碱化。因此，该反转运蛋白的纯化和表征对阐明信号转导具有重要意义。本研究将Na^+/H^+反转运蛋白与大鼠肾刷状缘膜辛基- β - d -葡萄糖吡喃苷溶解，并在卵磷脂酰胆碱脂质体中重组。用一种新的方法测定了反转运蛋白的活性。在含有pH敏感荧光染料异硫氰酸-葡聚糖荧光素（fitc -葡聚糖）的溶液中进行重构，pH为5.5。包封fitc -葡聚糖的重构囊泡在pH 8.0下孵育，通过监测染料的荧光变化来测定囊泡内pH （pHi）。观察到荧光强度的增加与pHi的增加相对应。H^+的增加速率取决于泡外Ne+的浓度，在150 mM葡萄糖酸钠存在时，H^+的外排被添加阿米洛利抑制。这些结果表明Na^+/H^+反转运蛋白在功能上被重构，其活性可以简单地通过使用fitc -葡聚糖来测定。为了了解麻醉的作用机制，我们还研究了局麻药对脂质体中Na^+/H^+反转运蛋白重构的影响。二布卡因和丁卡因减少了H^+的流出，而不影响Na^+梯度的存在。另一方面，10 μ m时利多卡因在存在向内Na^+梯度的情况下抑制H^+外流，表明局麻药与Na^+/H^+反转运体和/或其他Na^+转运体的直接相互作用可能是麻醉的触发因素。

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.