title: Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2).
abstract: 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 muM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter.PAT2 preferred substrates are L-alpha-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 alpha-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH(2) groups, as in beta-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.
authors.affiliation: Institute for Cell & Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kennedy
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Gatfield
authors.forename: John P
authors.initials: JP
authors.lastname: Winpenny
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
authors.forename: David T
authors.initials: DT
authors.lastname: Thwaites
mesh_terms: D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism*; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001622:Betaine / Q000378:metabolism; D001692:Biological Transport; D005260:Female; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011392:Proline / Q000378:metabolism; D011522:Protons; D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D011522:Protons; C474940:Slc36a2 protein, rat; D027981:Symporters; D001622:Betaine; D011392:Proline; D005998:Glycine