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

1. 为了更好地了解非洲爪蟾卵母细胞中pHR （halorhodopsin）的电致阴离子转运机制，我们建立了非洲爪蟾卵母细胞中N. pharaonis halorhodopsin （pHR）的表达体系。在该系统中，可以精确地测定阴离子输运引起的光诱导电流，从而分析输运过程的动力学。通过这种方法，我们能够证明通过pHR的Cl^-泵活性依赖于膜电位。在这种电压依赖性的基础上，我们首次证明了pHR的泵浦电流降至零时的V_R值代表了受激发的pHR分子将Cl^-泵入细胞2的本征离子动力。我们建立了过表达hPEPT1的HeLa细胞系（HeLa/hPEPT1），利用全细胞膜片钳技术测量了摄取和外排过程中的运输电流。在这里，我们描述了由于底物摄取后通过细胞外洗涤观察到的外排过程而产生的电流。基于这些数据，我们发现在外排过程中，二肽的疏水性参与了与hPEPT1的细胞内相互作用，从而打开了运输途径，导致“内向泄漏”电流采用电生理技术和摄取实验研究了人Na^+/单羧酸共转运体（hSMCT）在爪蟾卵母细胞中表达后的功能特征和底物特异性。我们已经确定了衬底的结构特征，这是它们与hSMCT相互作用的最低要求。根据与hSMCT相互作用的最小结构要求，我们还确定了与hSMCT相互作用最有效的先导化合物。

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.