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Lookup NU author(s): Dr David Kennedy,
Dr John Winpenny,
Professor David Thwaites
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Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrate-evoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2. Amino (and imino) acid transport via rPAT2 was pH-dependent, Na+-independent and electrogenic. At extracellular pH 5.5 (in Na+-free conditions) proline uptake was saturable (Km 172 ± 41 μM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter. PAT2 preferred substrates are L-α-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4- or 5-membered heterocyclic amino and imino acids such as 2-azetidine- carboxylate, proline and cycloserine, where both D- and L-enantiomers are transported. The major restrictions on transport are side chain size (the ethyl group of α-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH2 groups, as in β-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded. The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2.
Author(s): Kennedy DJ, Gatfield KM, Winpenny JP, Ganapathy V, Thwaites DT
Publication type: Article
Publication status: Published
Journal: British Journal of Pharmacology
Print publication date: 01/01/2005
ISSN (print): 0007-1188
ISSN (electronic): 1476-5381
Publisher: John Wiley & Sons Ltd.
PubMed id: 15644866
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