Foltz Martin, Boll Michael, Raschka Ladislav, Kottra Gabor, Daniel Hannelore
Molecular Nutrition Unit, Institute of Nutritional Sciences, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany.
FASEB J. 2004 Nov;18(14):1758-60. doi: 10.1096/fj.03-1387fje. Epub 2004 Sep 2.
Recently, the PAT family of proton-dependent amino acid transporters has been identified as a novel class of mammalian amino acid symporters. PAT1 and PAT2 members mediate electrogenic uptake of small, neutral amino acids and derivatives by cotransport of protons. Analysis of the structural requirements for substrate recognition by PAT1 identified that a free amino group in a substrate is not essential for recognition. We therefore hypothesized that PAT1 and its ortholog PAT2 may also be able to recognize and transport the homologous short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. We examined in Xenopus laevis oocytes whether the SCFAs interact with the transporter by employing flux studies, electrophysiology and intracellular pH recordings. SCFAs did not induce positive inward currents but inhibited glycine-induced transport currents. PAT-mediated uptake of radiolabeled proline was also dose-dependently reduced by SCFA and could be described by first order competition kinetics with apparent Ki-values for butyrate of 6.0 +/- 0.7 and 7.6 +/- 1.3 mM for PAT1 and PAT2, respectively. Acetate as well as propionate uptake was significantly enhanced in oocytes expressing PAT1 or PAT2. An electroneutral H+/SCFA symport mode was demonstrated by recording intracellular pH changes under voltage clamp conditions with rate constants for the initial intracellular acidification in the presence of SCFAs significantly increased in PAT-expressing oocytes. In conclusion, our data demonstrate that the PAT1 and PAT2 proteins are capable to transport selected SCFAs in an electroneutral and the homologous amino acids in an electrogenic mode and are therefore a paradigm for bifunctional solute carriers.
最近，质子依赖性氨基酸转运体的PAT家族已被确定为一类新型的哺乳动物氨基酸同向转运体。PAT1和PAT2成员通过质子共转运介导小的中性氨基酸及其衍生物的电驱动摄取。对PAT1识别底物的结构要求进行分析后发现，底物中的游离氨基对于识别并非必不可少。因此，我们推测PAT1及其直系同源物PAT2或许也能够识别并转运同源的短链脂肪酸（SCFAs），例如乙酸盐、丙酸盐和丁酸盐。我们在非洲爪蟾卵母细胞中，通过通量研究、电生理学和细胞内pH记录，检测了SCFAs是否与该转运体相互作用。SCFAs并未诱导正向内向电流，但抑制了甘氨酸诱导的转运电流。SCFAs也会剂量依赖性地降低PAT介导的放射性标记脯氨酸的摄取，并且可以用一级竞争动力学来描述，PAT1和PAT2对丁酸盐的表观Ki值分别为6.0±0.7 mM和7.6±1.3 mM。在表达PAT1或PAT2的卵母细胞中，乙酸盐和丙酸盐的摄取均显著增强。通过在电压钳制条件下记录细胞内pH变化，证实了一种电中性的H⁺/SCFA同向转运模式，在表达PAT的卵母细胞中，存在SCFAs时初始细胞内酸化的速率常数显著增加。总之，我们的数据表明，PAT1和PAT2蛋白能够以电中性模式转运选定的SCFAs，并以电驱动模式转运同源氨基酸，因此是双功能溶质载体的一个范例。
