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

Na^+/H^+逆向转运蛋白存在于多种细胞的质膜上，在调节细胞内pH值方面起重要作用。已知Na^+/H^+逆向转运蛋白可被多种刺激物（如生长因子）激活并引起细胞内碱化。因此，这种反向转运蛋白的纯化和表征对于阐明信号转导具有重要意义。在这项研究中，Na^+/H^+逆向转运蛋白用大鼠肾刷状缘膜的辛基-β-D-吡喃葡萄糖苷溶解，并在卵磷脂脂质体中重构。用一种新的方法测定了反向转运蛋白的活性。在含有pH敏感性荧光染料异硫氰酸荧光素-葡聚糖（FITC-葡聚糖）的溶液中在pH 5.5下进行重构。将包封FITC-葡聚糖的重构囊泡在pH 8.0下孵育，并通过监测染料的荧光变化来确定囊泡内pH（pHi）。观察到荧光强度的增加，其对应于pHi的增加。增加的速率依赖于囊外Ne+的浓度，在150 mM葡萄糖酸钠存在下，H^+的流出被阿米洛利抑制。这些结果表明，Na^+/H^+逆向转运蛋白是功能性重组的，而且其活性可以通过使用FITC-葡聚糖简单地测定。为了理解麻醉的机制，我们还研究了局部麻醉药对脂质体中重组的Na^+/H^+逆向转运蛋白的影响。地布卡因和丁卡因不依赖于Na^+梯度的存在而减少H^+流出。另一方面，10 μ M的利多卡因在存在内向Na^+梯度的情况下抑制H^+流出，表明局麻药与Na^+/H^+反向转运蛋白和/或其他Na^+转运蛋白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.