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

Abstract

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 substrateevoked 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 þ -independent and electrogenic. At extracellular pH 5.5 (in Na þ -free conditions) proline uptake was saturable (Km 172741 mM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter. 3 PAT2 preferred substrates are L-a-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-azetidinecarboxylate, 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 a-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 b-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.

Characterization of a blood-meal-responsive proton-dependent amino acid transporter in the disease vector, Aedes aegypti

“…AaePAT1 is a low affinity transporter with an ability to transport a number of different amino acids, including amino acids with small and aromatic side chains. The low affinity and low specificity of transport observed here is similar to that of mammalian PAT1 orthologues (Boll et al, 2003a;Boll et al, 2002;Chen et al, 2003a;Kennedy et al, 2005).…”

“…PAT1 has been identified in lysosomes and on the apical membrane of intestinal epithelial cells, where it is involved in amino acid absorption across the membrane (Sagne et al, 2001). PAT1 mRNA is found in all tissues, but PAT2 mRNA seems to be expressed in the lung, heart, kidney and muscle (Boll et al, 2003a;Boll et al, 2002;Broer, 2008;Kennedy et al, 2005).…”

“…The activity of this family of transporters is independent of Na + , K + and Cl -, but demonstrates dependence on pH, with amino acid uptake resulting in acidification of the cell (Abbot et al, 2006;Boll et al, 2002). Several PATs have been identified in vertebrates, and they fall into four PAT subclasses, but only PAT1 and PAT2 subclasses from human, mouse and rat have been characterized (Boll et al, 2003a;Boll et al, 2002;Chen et al, 2003a;Chen et al, 2003b;Kennedy et al, 2005;Sagne et al, 2001). PAT1 is a low affinity transporter with the ability to transport many small chain amino acids as well as γ-aminobutyric acid (Boll et al, 2003a;Boll et al, 2002).…”

Collectrin and ACE2 in renal and intestinal amino acid transport

“…were also described to transport imino acids and additionally glycine and β-alanine in intestine and kidney. [36][37][38][39][40] There is no knockout mouse model for SIT1 (slc6a20) yet.…”

SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

“…The pharmaceutically active amino acid derivatives that PAT1 can transport include the orally delivered antibiotic and antischizophrenic agent cycloserine (5), the orally active antihyperglycemic drug ␤-guanidinopropionic acid (6), the experimental anti-insomnia compound gaboxadol (7), the anti-epileptic vigabatrin (8), and the photodynamic therapy agent 5-aminolevulinic acid (9,10). PAT2-transported drugs include a number of neuroactive compounds, including cycloserine (an antischizophrenic), L-2-azetidine-carboxylate, pipecolic acid, nipecotic acid, and isonipecotic acid (11).…”