Kottra Gabor, Stamfort Adelmar, Daniel Hannelore
Molecular Nutrition Unit, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany.
J Biol Chem. 2002 Sep 6;277(36):32683-91. doi: 10.1074/jbc.M204192200. Epub 2002 Jun 24.
The capability for electrogenic inward transport of substrates that carry different net charge is a phenomenon observed in a variety of membrane-solute transporters but is not yet understood. We employed the two-electrode voltage clamp technique combined with intracellular pH recordings and the giant patch technique to assess the selectivity for bidirectional transport and the underlying stoichiometries in proton to substrate flux coupling for electrogenic transfer of selected anionic, cationic, and neutral dipeptides by the intestinal peptide transporter PEPT1. Anionic dipeptides such as Gly-Asp and Asp-Gly are transported in their neutral and negatively charged forms with high and low affinities, respectively. The positive transport current obtained with monoanionic substrates results from the cotransport of two protons. Cationic dipeptides can be transported in neutral and positively charged form, resulting in an excess transport current as compared with neutral substrates. However, binding and transport of cationic dipeptides shows a pronounced selectivity for the position of charged side chains demonstrating that the binding domain of PEPT1 is asymmetric, both in its inward and outward facing conformation. The simultaneous presence of identically charged substrates on both membrane surfaces generates outward and, unexpectedly, enhanced inward transport currents probably by increasing the turnover rate.
携带不同净电荷的底物进行电生性内向转运的能力是在多种膜 - 溶质转运体中观察到的一种现象，但目前尚未完全理解。我们采用双电极电压钳技术结合细胞内pH记录和巨膜片技术，来评估肠道肽转运体PEPT1对选定的阴离子、阳离子和中性二肽进行电生性转运时双向转运的选择性以及质子与底物通量偶联的潜在化学计量关系。阴离子二肽如甘氨酸 - 天冬氨酸（Gly - Asp）和天冬氨酸 - 甘氨酸（Asp - Gly）分别以中性和带负电荷的形式，以高亲和力和低亲和力进行转运。单阴离子底物产生的正向转运电流是由两个质子的共转运引起的。阳离子二肽可以以中性和带正电荷的形式进行转运，与中性底物相比会产生过量的转运电流。然而，阳离子二肽的结合和转运对带电侧链的位置表现出明显的选择性，这表明PEPT1的结合结构域在向内和向外的构象中都是不对称的。膜两侧同时存在带相同电荷的底物可能通过提高周转率产生外向且出乎意料地增强的内向转运电流。
