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

1 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. 2 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  μ M ), demonstrating that rPAT2 is, relative to PAT1, a high‐affinity transporter. 3 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. 4 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 CH 2 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. 5 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.British Journal of Pharmacology (2005) 144 , 28–41. doi: 10.1038/sj.bjp.0706029