Proton-coupled amino acid transport (PAT) systems in the apical membrane of epithelial cells of the small intestine and the renal tubule have been demonstrated by use of the human intestinal cell line Caco-2 and renal brush border membrane vesicles. The present thesis summarises the revelation of the molecular entity of the PAT system and provides deeper insights into this new mammalian family of proton/amino acid cotransporters. The identified family comprises four structural similar murine and human transport proteins (proton/amino acid transporter, PAT1 - 4), all consisting of around 500 amino acid residues with 11 predicted transmembrane spanning domains. Orthologous proteins of this family are found in lower and higher eukaryotes. The murine PATs exhibit a distinct mRNA expression pattern, with PAT1 found inter alia in epithelial cells of small intestine and kidney. Moreover, the PAT1 protein is localised in the brush border membrane of murine and human small intestine. After expression in Xenopus laevi oocytes PAT1 mediates proton-coupled, electrogenic transport of small and neutral amino acids such as Gly, Ala, and Pro, the corresponding D-isomers and of a variety of amino acid derivatives, e.g. GABA, betaine, and taurine. The apparent affinities for these substrates are in the range of 1 - 10 mM. Beside these physiological substrates PAT1 transports efficiently selected pharmacological compounds. As shown recently, PAT1 resembles in all features the functionally described PAT system in intestinal and renal epithelial cells. The transporter PAT2, expressed in the central nervous system, lung, heart, and brain exhibits similar transport characteristics as PAT1. However, PAT2 is in comparison to PAT1 a high affinity transporter, with 10 to 30- times higher affinities for the identical substrates (app. Km-values of 100 - 1000 µM) but has in general a narrower substrate spectrum. So far, PAT3 and PAT4 remain orphan transporters with no assigned function. Critical structural elements in aliphatic amino acid substrates of PAT1 and PAT2 were identified a) an non-substituted carboxyl-group, b) a small size of the amino acid side chain, and c) a short spacer distance between the charged amino and carboxyl groups. A free amino group within a substrate is not required. Additionally, short-chain fatty acids were shown to serve as substrates of PAT1 and PAT2. In contrast to amino acids, the short-chain fatty acids are transported in an electroneutral manner. Both transporters seems to bind proton and substrate in an ordered binding mode with the proton binding first. Functional differences in transport characteristics between PAT1 and PAT2 were observed in regard to the proton activation constants, suggesting that PAT2 transports at physiological pH values under proton-saturated conditions, whereas PAT1 can be regulated in transport activity by changes in extracellular pH, i.e. via a cooperation with the Na+/H+-exchanger NHE3. This novel family of proton/amino acid symporters takes an exceptional position within mammalian physiology.     «

Proton-coupled amino acid transport (PAT) systems in the apical membrane of epithelial cells of the small intestine and the renal tubule have been demonstrated by use of the human intestinal cell line Caco-2 and renal brush border membrane vesicles. The present thesis summarises the revelation of the molecular entity of the PAT system and provides deeper insights into this new mammalian family of proton/amino acid cotransporters. The identified family comprises four structural similar murine and...     »

Epithelzellen des Dünndarmes besitzen die Fähigkeit, kleine, neutrale Aminosäuren und deren Derivate im Ko-Transport mit Protonen aufzunehmen. In dieser Arbeit wird die molekulare Grundlage für diese Transportaktivität erstmals beschrieben und eine neue Familie von Protonen-Aminosäure-Transportern (PAT) in Säugetieren charakterisiert. Die PAT-Familie umfasst vier Mitglieder, die eine sehr hohe Ähnlichkeit in ihrer Aminosäuresequenz aufweisen. Die murinen Transporter zeigen auf mRNA-Ebene ein heterogenes Expressionsmuster. Das PAT1-Protein ist in der Membran des Dünndarmes der Maus lokalisiert. Auf funktioneller Ebene entsprechen die Charakteristika des klonierten PAT1 dem aus biochemischen Studien bekannten System PAT. PAT2 besitzt ähnliche Transporteigenschaften wie PAT1, weist aber höhere Affinitäten zu den identischen Substraten auf. Den Transportern PAT3 und 4 konnte noch keine Funktion zugeordnet werden. Die PAT-Familie nimmt eine Ausnahmestellung in der Säugetierphysiologie ein.     «

Epithelzellen des Dünndarmes besitzen die Fähigkeit, kleine, neutrale Aminosäuren und deren Derivate im Ko-Transport mit Protonen aufzunehmen. In dieser Arbeit wird die molekulare Grundlage für diese Transportaktivität erstmals beschrieben und eine neue Familie von Protonen-Aminosäure-Transportern (PAT) in Säugetieren charakterisiert. Die PAT-Familie umfasst vier Mitglieder, die eine sehr hohe Ähnlichkeit in ihrer Aminosäuresequenz aufweisen. Die murinen Transporter zeigen auf mRNA-Ebene ein het...     »