Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2)

David J Kennedy, Kelly M Gatfield, John P Winpenny, Vadivel Ganapathy, David T Thwaites

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)28-41
Number of pages14
JournalBritish Journal of Pharmacology
Volume144
Issue number1
DOIs
Publication statusPublished - Jan 2005

Keywords

  • Amino Acid Transport Systems/genetics
  • Amino Acid Transport Systems, Neutral/genetics
  • Amino Acids/metabolism
  • Animals
  • Betaine/metabolism
  • Biological Transport
  • Female
  • Glycine/metabolism
  • Hydrogen-Ion Concentration
  • Microinjections
  • Oocytes/metabolism
  • Patch-Clamp Techniques
  • Proline/metabolism
  • Protons
  • Rats
  • Stereoisomerism
  • Substrate Specificity
  • Symporters/genetics
  • Xenopus laevis

Fingerprint

Dive into the research topics of 'Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2)'. Together they form a unique fingerprint.

Cite this