Miyauchi Seiji, Abbot Emily L, Zhuang Lina, Subramanian Radhika, Ganapathy Vadivel, Thwaites David T
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA.
Mol Membr Biol. 2005 Nov-Dec;22(6):549-59. doi: 10.1080/09687860500421779.
Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H(+)/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5' and 3' untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na(+)-independent uptake of proline, glycine, l-alanine and alpha-(methylamino)isobutyric acid. Proline uptake was maximal at pH 5.0 (K(m) 2.2+/-0.7 mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na+ but in the presence of an inwardly directed H+ gradient. In the presence of Na+ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.
氨基酸的重吸收是肾近端小管的一项重要功能。先前已使用兔肾刷状缘膜囊泡（BBMV）测量了pH依赖性氨基酸转运。本研究的目的是确定这种pH依赖性摄取是否代表通过类似PAT1的转运系统进行的H⁺/氨基酸共转运。分离出兔PAT1 cDNA（2296bp，包括5'和3'非翻译区以及聚腺苷酸尾），其开放阅读框编码一个475个氨基酸的蛋白质（与人PAT1的同一性为92%）。在包括肾脏在内的所有研究组织中均发现了兔PAT1 mRNA。当在哺乳动物细胞系中异源表达时，兔PAT1介导脯氨酸、甘氨酸、L-丙氨酸和α-（甲基氨基）异丁酸的pH依赖性、Na⁺非依赖性摄取。脯氨酸摄取在pH 5.0时最大（Kₘ 2.2±0.7 mM）。在兔肾BBMV中检测到一个具有相同特征（离子依赖性、底物特异性）的转运系统，在无Na⁺但存在内向H⁺梯度的情况下观察到超调现象。在存在Na⁺且PAT1转运功能受到抑制的条件下，检测到第二个脯氨酸摄取系统，其功能特征与亚氨基酸系统相似。兔PAT1在哺乳动物细胞或肾BBMV中的功能特征表明，PAT1是脯氨酸、甘氨酸和羟脯氨酸的低亲和力转运体，据信在亚氨基甘氨酸尿症患者中存在缺陷。
