| Budget Amount *help |
¥4,020,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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.
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