Amino acid and drug transport via members of the SLC36 family are usually measured by uptake of [3H] or [14C] labelled ligands (e.g. Thwaites et al., 1993a, 1995c; Sagnéet al., 2001; Boll et al., 2002; Chen et al., 2003a; 2003b; Wreden et al., 2003; Anderson et al., 2004). If a compound is unavailable in radiolabelled form, evidence for its transport via the carrier can be determined by the relative ability of the compound to cis-inhibit (when present in the ipsilateral compartment) and trans-stimulate (when present at the contralateral side of the membrane) the uptake of a standard substrate (e.g. Thwaites et al., 1995c; 2000; Boll et al., 2002; 2003a; Anderson et al., 2004, 2010; Foltz et al., 2004b; 2005; Kennedy et al., 2005; Metzner et al., 2006; Edwards et al., 2011).
Both PAT1 and PAT2 transport zwitterionic amino acids in a 1:1 stoichiometry with a H+ (Thwaites et al., 1994; Boll et al., 2002; Wreden et al., 2003), meaning that both carriers are rheogenic (i.e. able to generate current) and membrane potential sensitive. The movement of the coupling ion (in this case a H+) has been used to measure PAT1 function at the apical membrane of human intestinal epithelial Caco-2 cell monolayers where PAT1-mediated H+-influx has been measured as a decrease in pHi in cells loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (e.g. Thwaites et al., 1993a; 1995c; 2000; Anderson et al., 2004; Abbot et al., 2006); an increase in inward short-circuit current in voltage-clamped Caco-2 cell monolayers in Ussing chambers (e.g. Thwaites et al., 1993a,b; 1994; 1995b,c; 2000); a change in membrane potential using a red membrane potential-sensitive dye (Metzner et al., 2005, 2006). In cultured isolated rat hippocampal neurones, PAT1 function has been detected using pH-sensitive green fluorescent protein (Wreden et al., 2003).
To allow detailed functional characterization of either PAT1 or PAT2 in isolation, the transporters have been expressed heterologously either in mammalian cell lines (e.g. COS-7, HRPE or HeLa cells) (Sagnéet al., 2001; Chen et al., 2003a; 2003b; Wreden et al., 2003; Miyauchi et al., 2005) or in Xenopus laevis oocytes (e.g. see Boll et al., 2002; 2003a; Chen et al., 2003b; Wreden et al., 2003; Anderson et al., 2004, 2009, 2010; Foltz et al., 2004a, 2005; Kennedy et al., 2005; Metzner et al., 2005; 2009; Abbot et al., 2006; Edwards et al., 2011). Expression in X. laevis oocytes allows detailed kinetic analysis of transporter function by measurement of substrate-induced inward current (H+ movement) using electrophysiological techniques such as two-electrode voltage-clamp (TEVC) or giant patch clamp (Boll et al., 2002; 2003a; Wreden et al., 2003; Anderson et al., 2004; 2010; Foltz et al., 2004a; 2005; Kennedy et al., 2005). H+ movement has also been measured using H+-sensitive intracellular electrodes in oocytes (Boll et al., 2002; Foltz et al., 2004a) or BCECF in PAT1-transfected HeLa cells (Wreden et al., 2003).
PAT1 transport has been characterized using cDNA clones from human, rat, mouse and rabbit, and no marked differences in transport (substrate specificity or substrate affinity) have been observed between these orthologues. PAT1 is a H+-coupled, pH-dependent, low-affinity transporter (Km approximately 1–10 mM) of small, unbranched, zwitterionic α-, β- and γ-amino and imino acids; N-methylated amino acids; and heterocyclic amino acids containing four- to six-membered rings (Thwaites and Anderson, 2007a,b). Substrates include glycine, proline, alanine, trans-4-hydroxy-proline, β-alanine, taurine, sarcosine, betaine and GABA; the d-amino acids d-proline, d-alanine, d-cysteine and d-serine; and the amino acid derivatives α-(methylamino)isobutyric acid (MeAIB), α-aminoisobutyric acid (AIB) and β-aminobutyric acid (β-ABA) (see Table 1) (Sagnéet al., 2001; Boll et al., 2002; 2003a; Chen et al., 2003a; Wreden et al., 2003; Anderson et al., 2004; 2009; Miyauchi et al., 2005; Abbot et al., 2006). This substrate specificity is identical to that described for system PAT at the apical membrane of human Caco-2 cell monolayers (Thwaites et al., 1993a; 1994; 1995b,c; Thwaites and Stevens, 1999; Anderson et al., 2004, 2009; Metzner et al., 2004, 2006) and the imino acid carrier at the luminal surface of the rat small intestine (Newey and Smyth, 1964; Munck, 1966; Wiseman, 1968; Daniels et al., 1969a,b; Thompson et al., 1970; De la Noüe et al., 1971; Munck et al., 1994; Anderson et al., 2004; Iñigo et al., 2006). A comprehensive review of the relationship between the imino acid carrier, system PAT and PAT1 can be found in Thwaites and Anderson (2007a). The most detailed investigations of the substrate specificity of this carrier can be found in Thwaites et al. (1995c), Boll et al. (2003a) and Metzner et al. (2006). In addition to rheogenic H+/zwitterionic amino acid transport, PAT1 can also function as an electroneutral transport system for H+ and fatty acids including acetate, propionate and butyrate (Foltz et al., 2004a).
The low-affinity, high-capacity, transport characteristics and relatively broad substrate specificity identify PAT1 as a potential pathway for transport of orally delivered hydrophilic amino acid-based drugs across the luminal surface of the human small intestinal wall, the initial barrier to effective drug delivery (Figure 1). In particular a number of GABA- and proline-related compounds have been identified as PAT1 substrates (Ranaldi et al., 1994; Thwaites et al., 1995a; 2000; Anderson et al., 2004; Metzner et al., 2004; 2006; 2009; Abbot et al., 2006; Larsen et al., 2008; 2009; 2010). PAT1 substrates with therapeutic, or potential therapeutic, value include nipecotic acid, guvacine and THPO (GAT transporter inhibitors) (Thwaites et al., 2000; Anderson et al., 2004; Abbot et al., 2006; Larsen et al., 2008); isonipecotic acid, muscimol and gaboxadol (GABAA receptor agonists) (Thwaites et al., 2000; Anderson et al., 2004; Larsen et al., 2008; 2009; 2010); vigabatrin (a GABA-transaminase inhibitor) (Abbot et al., 2006); 1-aminocyclopropanecarboxylic acid (a partial agonist for the NMDA receptor) (Thwaites et al., 2000); 3-amino-1-propanesulphonic acid (an anticraving agent) (Thwaites et al., 2000; Metzner et al., 2004); l-azetidine-2-carboxylic acid and 3,4-dehydro-d,l-proline (inhibitors of collagen synthesis) (Metzner et al., 2004, 2006); l-cycloserine (an inhibitor of cancer cell growth in vitro) (Anderson et al., 2004; Metzner et al., 2006); 5-aminolevulinic acid (used in photodynamic therapy, fluorescent diagnosis and fluorescent-guided resection of cancer) (Anderson et al., 2010); d-serine (used to treat schizophrenia) (Thwaites et al., 1995a,c; Boll et al., 2002; Chen et al., 2003a); d-cycloserine (used as an antibiotic and in the treatment of schizophrenia) (Ranaldi et al., 1994; Thwaites et al., 1995a; Anderson et al., 2004); betaine (used in the treatment of homocystinuria) (Thwaites et al., 1995c; Boll et al., 2003a); and β-guanidinopropionic acid (an anti-hyperglycaemic agent) (Metzner et al., 2009) (Figure 1; Table 1).
Figure 1. The H+-coupled amino acid transporter PAT1 (SLC36A1) can transport a number of compounds with potential therapeutic value (shown in blue). Several analogues of tryptophan such as 5-hydroxy-l-tryptophan (shown in green) are non-transported inhibitors of PAT1.
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The compounds listed above are all transported substrates of PAT1 and will thus compete for transport. In contrast, Brandsch and colleagues identified that l-tryptophan and the derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin and indole-3-propionic acid are non-transported, competitive inhibitors of PAT1 (Ki 0.9–6.1 mM) (Figure 1) (Metzner et al., 2005). 5-Hydroxy-l-tryptophan has been used to identify the relative contribution of PAT1 and the di/tripeptide transporter PepT1 (SLC15A1) to uptake of 5-aminolevulinic acid at the apical membrane of human intestinal Caco-2 cell monolayers (Anderson et al., 2010). In addition, l-tryptophan and 5-hydroxy-l-tryptophan have been used in vivo in studies in dogs and rats, respectively, to reduce gaboxadol absorption (Larsen et al., 2009, 2010). These non-transported inhibitors are likely to prove highly useful tools to aid in the identification of the relative contribution of PAT1 to substrate transport in membranes and tissues expressing multiple transporters with overlapping substrate specificity. However, the absolute value of the compounds may be restricted to some degree as the affinity for PAT1 is relatively low and, as yet, it is not known how many other carriers may also be inhibited by these compounds. For example, another tryptophan derivative, α-methyl-d,l-tryptophan, acts as a relatively high affinity (IC50 250 µM) non-transported inhibitor of the amino acid transporter ATB0,+ (SLC6A14) (Karunakaran et al., 2008).
PAT1 can be considered as a low affinity multipurpose transporter with substrate specificity that overlaps with many of the SLC6 family of Na+- and Cl--dependent neurotransmitter transporters including GAT1 (SLC6A1), GlyT2 (SLC6A5), TauT (SLC6A6), PROT (SLC6A7), CT1 (SLC6A8), GlyT1 (SLC6A9), GAT3 (SLC6A11), BGT1 (SLC6A12), GAT2 (SLC6A13), ATB0,+ (SLC6A14), SBAT1 (SLC6A15), NTT4 (SLC6A17), XT2 (SLC6A18), B0AT1 (SLC6A19) and SIT1 (SLC6A20). In general, the SLC6 transporters are highly selective and have a much higher affinity for their substrates compared to PAT1. Therefore, we can speculate that the role of PAT1 is maybe to function as a high-capacity route for mass transfer of substrates in tissues where the high-affinity SLC6 carriers might be saturated. Evidence to date suggests that PAT1 is not generally co-expressed at the same membrane surfaces as SLC6 members except at the luminal surface of the small intestine where TauT, CT1, ATB0,+, B0AT1 and SIT1 are expressed to varying degrees. The relative roles of PAT1 and other carriers in neutral amino acid transport in the intestine and kidney are reviewed elsewhere (Thwaites and Anderson, 2007a; Bröer, 2008a,b).
PAT2, like PAT1, is a rheogenic H+-coupled, pH-dependent, transporter of small, unbranched, dipolar amino and imino acids but has a higher affinity (Km 0.1–0.7 mM) than PAT1 and has a relatively restricted substrate profile (Table 1). PAT2 substrates include glycine, alanine, l- and d-proline, trans-4-hydroxy-proline, sarcosine and the amino acid derivative MeAIB (Boll et al., 2002; Chen et al., 2003b; Rubio-Aliaga et al., 2004; Foltz et al., 2004b; Kennedy et al., 2005; Bröer et al., 2008; Edwards et al., 2011). Like PAT1, PAT2 also transports amino acids containing a free carboxyl group and small side chain but has a preference for the amino group in the α position, is more selective for l- over d-enantiomers and is less tolerant of N-methylation (Foltz et al., 2004b; Kennedy et al., 2005). The Km values estimated from transport measurements using the mouse, rat or human PAT2 transporters are almost identical. PAT2 transports a number of heterocyclic compounds with four- or five-membered ring structures including some with potential in therapy such as d-cycloserine, l-cycloserine, l-azetidine-2-carboxylic acid and 3,4-dehydro-d,l-proline, all mentioned above (Table 1) (Kennedy et al., 2005; Edwards et al., 2011). Like PAT1, PAT2 can also function in an electroneutral mode whereby it cotransports a H+ and a short-chain fatty acid (such as acetate, propionate or butyrate) but with relatively low affinity (Foltz et al., 2004a). As observed with PAT1, a number of tryptophan derivatives can also inhibit PAT2 in a non-transported manner, with 5-hydroxy-l-tryptophan and α-methyl-d,l-tryptophan having the greatest affinities (IC50 1.6 and 3.5 mM respectively) (Edwards et al., 2011). Although relatively low affinity inhibitors, these two tryptophan derivatives should help discriminate between PAT1 and PAT2 transport, as 5-hydroxy-l-tryptophan has a preference for PAT1, whereas the two compounds inhibit PAT2 with similar potency (Edwards et al., 2011). The most detailed investigations of the substrate specificity of PAT2 can be found in Boll et al. (2002), Foltz et al. (2004b), Kennedy et al. (2005) and Edwards et al. (2011).
In a similar manner to PAT1 in the small intestine, the function of PAT2 in the renal proximal tubule is likely to overlap to some extent with members of the SLC6 family including XT2 (SLC6A18), B0AT1 (SLC6A19) and SIT1 (SLC6A20), all of which are likely to function in the reabsorption of glycine and imino acids from the renal filtrate.