title: Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2).
abstract: The H(+)-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[(3)H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC(50) 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.
authors.affiliation: Epithelial Research Group, Institute for Cell & Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Noel
authors.initials: N
authors.lastname: Edwards
authors.forename: Catriona M H
authors.initials: CM
authors.lastname: Anderson
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Gatfield
authors.forename: Mark P
authors.initials: MP
authors.lastname: Jevons
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
authors.forename: David T
authors.initials: DT
authors.lastname: Thwaites
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000379:methods; D020128:Inhibitory Concentration 50; D008956:Models, Chemical; D009865:Oocytes / Q000378:metabolism; D010641:Phenotype; D011392:Proline / Q000737:chemistry; D051381:Rats; D027981:Symporters / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014982:Xenopus laevis / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; C474940:Slc36a2 protein, rat; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D014364:Tryptophan; D011392:Proline