A new approach to investigate on electrogenic transporters with Xenopusoocyte expression system

1.光驱动氯离子泵（盐视紫红质）对氯离子的转运机制为了更好地了解非洲爪蟾卵母细胞中pharaonis halorhodopsin（pHR）介导的产电阴离子转运机制，在该系统中，可以精确地测定阴离子转运的光诱导电流，从而分析转运过程的动力学。通过这种方法，我们能够证明通过pHR的Cl^-泵活性取决于膜电位。基于这种电压依赖性，我们首次证明了pHR泵电流降为零时的V_R值代表了受激pHR分子将Cl^-泵入细胞的本征离子动力.人寡肽转运蛋白（hPEPT 1）对二肽的转运机制我们建立了过表达hPEPT 1的HeLa细胞系（HeLa/hPEPT 1），用全细胞膜片钳技术测定了该细胞的摄取和外排转运电流。在这里，我们的特点是目前由于外排过程中观察到的底物摄取后的细胞外清洗。基于这些数据，我们发现二肽的疏水性参与了在流出过程中与hPEPT 1的细胞内相互作用，这可能会打开导致“内向漏”电流的转运途径。3人Na^+/单羧酸协同转运蛋白（hSMCT）的底物识别机制通过电生理技术和摄取研究确定了在非洲爪蟾卵母细胞中表达后的人SMCT的功能特性和底物特异性。我们已经确定了底物中的结构特征，这些结构特征对于它们与hSMCT的相互作用是最低限度需要的。根据与hSMCT相互作用的最低结构要求，我们还鉴定了与hSMCT最有效相互作用的先导化合物。

1. The transport mechanism of chloride ion by the light-driven chloride ion pump (halorhodopsin (HR))We have established a N. pharaonis halorhodopsin (pHR) expression system in Xenopus laevis oocytes to gain a better insight into the mechanism of the electrogenic anion transport via pHR. In this system, the photo-induced currents due to anion transport could be determined precisely to analyze the kinetics of the transport process. With this approach, we were able to demonstrate that the Cl^- pump activity via pHR is dependent on membrane potential. On the basis of this voltage-dependency, we show for the first time that the V_R value, at which the pump current by pHR is reduced to zero, represents the intrinsic ion motive force of the excited pHR molecule to pump Cl^- into cell.2. The transport mechanism of dipeptides by human oligopeptide transporter (hPEPT1)We have established a HeLa cell line over-expressing hPEPT1 (HeLa/hPEPT1), in which we measured the transport currents due to uptake and the efflux process using the whole-cell patch-clamp technique. Here, we have characterized the current due to the efflux process observed by an extracellular washing after substrate uptake. Based on these data, we found that the hydrophobicity of the dipeptide was involved in the intracellular interaction with hPEPT1 during an efflux process, which could open the transport pathway causing the 'inward leak' current.3 The mechanism of substrate recognition by human Na^+/ monocarboxylate co-transporter (hSMCT)Functional characteristics and substrate specificity of human SMCT after expression in Xenopus lavevis oocytes were determined by electrophysiological techniques and uptake studies. We have identified the structural features in substrates, which are required minimally for their interactions with hSMCT. According to the minimal structural requirements for interactions with hSMCT, we have also identified the lead compound, which interacts with hSMCT most potently.