The FASEB Journal Research Communication

Serotonin, L-tryptophan, and tryptamine are effective inhibitors of the amino acid transport system PAT1
Linda Metzner,* Gabor Kottra, Klaus Neubert, Hannelore Daniel, and Matthias Brandsch*,1
*Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenberg, Halle, Germany; Molecular Nutrition Unit, Center of Life and Food Science, Technical University of Munich, Freising-Weihenstephan, Germany; and the Institute of Biochemistry, Department of Biochemistry/Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle, Germany The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of L- and D-proline, Lalanine, glycine, taurine, D-serine, GABA, and many other related compounds and drugs. Here we describe the novel nding that L-tryptophan and its derivatives tryptamine, 5-hydroxy-L-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H -dependent 3 L-[ H]proline uptake into Caco-2 cells with inhibition constants (Ki) of 0.9 to 6.1 mM. Uptake of L-[3H]tryptophan into Caco-2 cells on the other hand was not inhibited by L-proline. Whereas PAT1 substrates produced signicant changes in a membrane potential assay for electrogenic transport in Caco-2 cells, Ltryptophan, tryptamine, and 5-hydroxy-L-tryptophan failed to alter membrane voltage. When PAT1 was expressed in Xenopus laevis oocytes and analyzed by the two-electrode voltage clamp technique, glycine elicited high inward currents that were dependent on membrane potential but no currents were observed with L-tryptophan, tryptamine, 5-hydroxy-L-tryptophan, or serotonin. Although not transported electrogenically by PAT1, L-tryptophan and its derivatives inhibited glycineevoked currents dose-dependently. We conclude that serotonin, L-tryptophan, and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers naturally occurring inhibitors that may alter the transport function of PAT1.Metzner, L., Kottra, G., Neubert, K., Daniel, H., Brandsch, M. Serotonin, L-tryptophan, and tryptamine are effective inhibitors of the amino acid transport system PAT1. FASEB J. 19, 1468 1473 (2005)
ABSTRACT

Key Words: membrane transport proton gradient nontransported inhibitors Xenopus laevis oocytes Caco-2 cells The proton-coupled amino acid transporters (PAT) constitute the recently identied SLC36 family of mammalian membrane transporters (1). The rst member was cloned as the lysosomal amino acid transporter 1 (LYAAT1) from rat brain (2) and subsequently PAT1 (orthologous to LYAAT1) and PAT2 were identied
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from mouse intestine and embryonic tissue (3). In 2003, the human PAT1 was cloned and immunolocalization studies revealed its presence in the apical membrane of intestinal Caco-2 cells (4). The transporter was nally shown to be the protein entity of the H /amino acid symport system functionally described rst 10 years ago (4 9). The PAT1-mRNA is found not only in intestine but also with lower expression levels in brain, colon, liver, lung, placenta, and testis (35). The PAT proteins mediate electrogenic uphill transport of substrates energized by a transmembrane electrochemical proton gradient. The primary substrates for hPAT1 are amino acids such as glycine, l-proline, and l-alanine. PAT1 appears to represent the major route by which small, unbranched, neutral amino and imino acids are absorbed after intestinal protein digestion (10). The PAT proteins may also have a pharmacological importance as they can transport drugs effective in treating affective disorders, homocystinuria, convulsions, and cancer (10). An unusual feature of the PAT system is that it also transports d-amino acids such as d-serine and d-cycloserine with afnities similar or even higher than those for glycine or l-proline. PAT proteins also transport GABA and important osmolytes such as betaine and taurine (3, 10, 11). Because PAT1 could serve as an oral drug delivery system, several studies have focused on the carriers substrate selectivity. We recently reported that hPAT1 in Caco-2 cells accepts numerous therapeutically relevant l-proline derivatives shown to prevent procollagen from folding into a stable triple-helical conformation such as 3,4-dehydro-d,l-proline, cis-4-hydroxy-l-proline, l-azetidine-2-carboxylic acid, and other structures (12). Critical recognition criteria are the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated (13). A bifunctional transport mode of PAT1 (and PAT2) was recently described by its capability for
1 Correspondence: Membrane Transport Group, Biozentrum of the Martin-Luther-University Halle-Wittenberg, Weinbergweg 22, D-06120 Halle, Germany. E-mail: matthias. brandsch@biozentrum.uni-halle.de doi: 10.1096/fj.05-3683com

0892-6638/05/0019-1468 FASEB

transport of short chain fatty acids such as acetate, propionate, and butyrate in an electroneutral mode and transport of the homologous amino acids in an electrogenic mode (14). In the present study we report the novel nding that l-tryptophan and the biogenic amines tryptamine and serotonin are recognized by PAT1 with afnities similar or even higher than those of the prototype substrates. However, they are not transported by hPAT1 and therefore may represent physiological inhibitors of the PAT proteins.

Xenopus laevis oocytes expressing mPAT1 and electrophysiology Female X. laevis were purchased from Nasco (Fort Atkinson, WI, USA). Surgically removed oocytes were separated by collagenase treatment and handled as described previously (18). Individual oocytes were injected with 10 nL of RNA solution containing 10 ng of mPAT1 cRNA. All electrophysiological measurements were performed after incubating oocytes for 2 4 days at 18C in a buffer composed of 88 mM NaCl, 1 mM KCl, 0.82 mM MgCl2 , 0.41 mM CaCl2 , 0.33 mM Ca(NO3)2 , 2.4 mM NaHCO3 , and 10 mM MES/Tris at pH 6.5 (modied Barth solution). To characterize the response current (I) in oocytes expressing mPAT1, the two-electrode voltage clamp technique was applied (18). Oocytes were placed in an open chamber and continuously superfused with Barth solution or with solutions also containing the compounds to be studied. Oocytes were voltage-clamped at 60 mV using a TEC-03 amplier (npi Electronic, Tamm, Germany), and currentvoltage (IV) relations were measured immediately before and 20 30 s after substrate application in the potential range of 140 to 60 mV. The current generated by the substrate transport at a given membrane potential was calculated as the difference of the currents measured in the presence and the absence of substrate. Data analysis Results are given as means se (n 4 8). Nonlinear regression analysis, calculation of inhibition constants (Ki) from IC50 values, and statistical analysis were done as described (12, 15, 18).

MATERIALS AND METHODS

Materials The intestinal cell line Caco-2 was obtained from the German Collection of Microorganisms and Cell Cultures. l-[3H]Proline (specic activity 43 Ci/mmol) and l-[3H]tryptophan (specic activity 27 Ci/mmol) were supplied by Amersham International (Arlington Heights, IL, USA). Cell culture reagents were purchased from Invitrogen (San Diego, CA, USA). Amino acids, biogenic amines, and derivatives were from Sigma-Aldrich (St. Louis, MO, USA). l-Tryptophan was also purchased from Roth (Karlsruhe, Germany). Culture of Caco-2 cells and L-[3H]proline uptake measurements Caco-2 cells were cultured (passages 6-63) in minimum essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acid solution, and gentamicin (45 g/mL) as described earlier (12, 15, 16). Cells at 80 90% density were released by trypsinization and subcultured in 35 mm disposable Petri dishes and 96-well plates. Medium was replaced the day after seeding, every 2 days, and the day before the experiment. With a starting cell density of 0.8 106 cells per dish, cultures reached conuence within 24 h. Uptake was measured on the 7th day after seeding. Uptake of l-[3H]proline was measured as described previously (12), using uptake buffer (1 mL) containing 25 mM MES/Tris (pH 6.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 10 nM l-[3H]proline, and unlabeled inhibitors at increasing concentrations. Uptake of l-[3H]tryptophan (10 nM) was measured under identical conditions. After 10 min incubation, cells were quickly washed four times with ice-cold buffer, solubilized, and prepared for liquid scintillation spectrometry. Protein was measured according to the procedure of Bradford. Membrane potential assay measurements Caco-2 cells subcultured as described above for 7 days but in 96-well plates at a starting density of 0.8 105 cells/well were washed twice with uptake buffer (pH 6.0) and incubated with 50 L red membrane potential (MP) dye (Molecular Devices Corp., Munich, Germany) per well for 60 min at 37C as established by Faria et al. (17). After that, compounds solved in 200 L uptake buffer for a nal concentration of 10 mM were added. Fluorescence was measured immediately at wavelengths of 530 nm (excitation) and 570 nm (emission) at a FLUOstar Galaxy (BMG Labtechnologies Offenburg, Germany).
SEROTONIN, L-TRYPTOPHAN ARE PAT1 INHIBITORS

RESULTS AND DISCUSSION Interaction of amino acids and biogenic amines with hPAT1 in Caco-2 cells In a series of experiments to elucidate the substrate specicity and pharmacological relevance of hPAT1, we determined the afnity of amino acids, biogenic amines, and their derivatives for interaction with hPAT1 expressed constitutively in Caco-2 cells (4, 6, 7, 12). In competition assays with l-[3H]proline as a prototype substrate of PAT1, we unexpectedly observed that l-tryptophan, 5-hydroxy-l-tryptophan, tryptamine, serotonin, and indole-3-propionic acid (10 mM) inhibited l-[3H]proline uptake by 52 to 70% (Table 1). In contrast, neither l-lysine or l-leucine (Table 1) nor l-phenylglycine, l-phenylalanine, l- and d-valine, ltyrosine, l-isoleucine, and many other amino acids (data not shown) altered signicantly l-[3H]proline inux. The effect of tryptophan was stereospecic for the l-conguration: d-tryptophan inhibited protoncoupled l-[3H]proline uptake only by 5% at a concentration of 10 mM and by 7% at 30 mM, whereas l-tryptophan inhibited uptake by 52 and 74% at concentrations of 10 and 30 mM, respectively. Inhibition of l-[3H]proline transport occurred in a dose-dependent manner (Fig. 1A, B) with apparent Ki values (Table 1) between 0.9 and 6.1 mM. l-Tryptophan and its derivatives serotonin, tryptamine, and indole-3-propionic acid
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TABLE 1. Transport properties of amino acids and biogenic amines for interaction with PAT1a
% Uptake L-[3H]proline Substrate/inhibitor L-[3H]tryptophan Caco-2 Cells Ki (mM) for L-[3H]Pro uptake

Fluorescence signal (%)

% I Gly

% Inhibition of I Gly

X. laevis oocytes

l-Proline Glycine GABA l-Tryptophan d-Tryptophan 5-OH-L-Tryptophan Indole-3-propionic acid Tryptamine Serotonin l-Lysine l-Leucine

32 56 34 48 95 30 37 30 45 99 102

4 2 3 4 7 1 3 1 3 3 4

98 2

9 1

1.8 5.0 4.0 4.7 0.9 4.6 6.1 5.7

0.2 0.6 0.3 0.3 0.1 0.3 0.4 0.6

100 98 102 23 32 13 18 12 23

2 3 2 3 2 5 6 5 2

95 4 100 51 1* 0 0 2 1 1 1 3 1 2 1 2 1 3 1 6 4

49 5 88 40 76 82 37 5

4 3 1 3 6 7 4 4

a Uptake of L-[3H]proline and L-[3H]tryptophan (10 nM) in Caco-2 cells was measured at pH 6.0 for 10 min in the absence ( 100%) or presence of unlabeled compounds (10 mM). Ki values were calculated from curves shown in Fig. 1 A, B. For measuring uorescence signal changes, concentrations of 10 mM (pH 6.0) were used. The signal elicited by 10 mM L-proline was set 100%. Signals below 30% were considered as not signicant. Percent IGly in oocytes was taken from the recordings of the I-V relationships representing the current evoked by 20 mM of the compounds and compared to that generated by 20 mM glycine at a membrane potential of 60 mV. The afnity constant (Kt) of glycine uptake at PAT1 expressed in oocytes is 7.0 0.7 mM (3). Inhibition of IGly was calculated from the currents generated by 5 mM glycine applied alone and together with 20 mM of the compound under test. Values are means se (n 4 8, : not determined, * value from ref 13).

therefore display afnities similar to those of the classical hPAT1 substrates such as l-proline, GABA, or glycine. 5-Hydroxy-l-tryptophan possesses an apparent Ki of 0.9 mM representing the highest afnity reported so far for any of the PAT1 substrates/ligands. We then determined the kinetics of l-proline uptake into Caco-2 cells at pH 6.0 in a concentration range of 0.520 mM in the absence or presence of 7 mM of l-tryptophan. In the absence of l-tryptophan, the Michaelis-Menten constant, Kt, for l-proline uptake was 1.4 0.5 mM and the maximal transport velocity, Vmax, was 78 13 nmol mg protein1/10 min. The corresponding parameters obtained in the presence of l-tryptophan were Kt 3.6 0.7 mM and Vmax 107 13 nmol mg protein1/10 min. The presence of l-tryptophan at a concentration close to its Ki value increased the Kt value of l-proline uptake 2.6-fold whereas the Vmax was not altered signicantly. This indicates that l-tryptophan inhibits hPAT1-mediated l-proline uptake in a competitive manner. To assess whether l-tryptophan is also transported by hPAT1, we used a membrane potential assay (17) and investigated whether the various amino acids and derivatives provided at 10 mM concentration induce measurable and signicant changes in membrane potential (MP) that should accompany electrogenic substrate driven proton inux with a 1:1 H ux coupling stoichiometry. As expected, l-proline, GABA, and glycine elicited maximum signals (Table 1) but also d-proline, d-alanine, taurine, l- and d-cyloserine, and l-azetidine-2-carboxylic acid produced 100% changes in MP (data not shown). Most important, signals elicited by serotonin, l- and dtryptophan, 5-hydroxy-l-tryptophan, and tryptamine were negligible suggesting that l-tryptophan, serotonin, and the other compounds are not transported electrogenically into Caco-2 cells. The afnity constants
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of these compounds are very similar or even lower than those of l-proline, GABA, glycine, d-proline, and l-azetidine-2-carboxylic acid (12), which did produce a 90 100% signal in the MP assay. Hence, using a 10 mM concentration is sufcient in this assay to differentiate substrates from inhibitors. The lack of transport mediated changes in MP despite the strong inhibition of substrate (l-proline) uptake could represent an inhibitor function or transport of the compounds in an electroneutral mode. To distinguish between these possibilities, we determined the uptake of l-[3H]tryptophan (10 nM) into Caco-2 cells at pH 6.0 and pH 7.5 in the absence and the presence of Na and excess amounts of unlabeled l-tryptophan or l-proline. The inux of l-[3H]tryptophan was neither H - nor Na -dependent. But unlabeled l-tryptophan at a concentration of 20 mM almost completely inhibited tracer l-tryptophan uptake (2.52 0.14 pmol mg protein1/10 min to 0.05 0.01 pmol mg protein1/10 min) whereas l-proline (20 mM) had no effect on l-[3H]tryptophan inux (remaining uptake: 2.46 0.02 pmol mg protein1/10 min). Hence, l-tryptophan strongly inhibited l-proline uptake but l-proline did not inhibit l-tryptophan inux, which allowed the conclusion that l-tryptophan is not transported by PAT1, not even in an electroneutral manner, leaving l-tryptophan as a nontransported inhibitor of PAT1. Interaction of amino acids and biogenic amines with mPAT1 expressed in X. laevis oocytes In Caco-2 cells, as in normal intestinal epithelial cells, at least eight different apical transport systems with, in some cases, overlapping substrate specicity, contribute
METZNER ET AL.

The FASEB Journal

pounds. However, similar to the Caco-2 cell model, the voltage-dependent inward currents induced by glycine (IGly) were dose-dependently reduced by increasing concentrations of l-tryptophan (Fig. 2B), again sug-

Figure 1. Inhibition of l-[3H]proline uptake in Caco-2 cells. Uptake of l-[3H]proline (10 nM, pH 6.0) was measured at increasing concentrations of unlabeled amino acids, biogenic amines, and derivatives (A, B). l-[3H]Proline uptake measured in the absence of inhibitors (667.1 74.1 fmol mg protein1/10 min) was taken as 100%. Values are means se, n 4.

to total amino acid uptake. In general, l-proline is transported at an enterocyte, e.g., by the amino acid transport systems PAT1, the recently cloned imino carrier SIT1 (19), ASCT1, and ATA2. In Caco-2 cells, under our conditions we found no evidence for an apical l-proline transporting system other than PAT1 (12) and neither did Thwaites group (6 8, 10, 11). Chen et al (4). performed a very detailed and technically comprehensive study regarding this question and also attributed apical l-proline transport to hPAT1. Nonetheless, to demonstrate unequivocally that it is the PAT1 that is specically inhibited by l-tryptophan and its derivatives, we determined amino acid-induced inward currents by the two-electrode voltage clamp technique in oocytes expressing mPAT1 (1, 3, 13, 14). As shown in Fig. 2A, glycine and l-proline (20 mM) caused high inward currents whereas l-leucine, l-tryptophan, indole-3-propionic acid, 5-hydroxy-l-tryptophan, tryptamine, and serotonin failed to evoke any currents, excluding the electrogenic transport of these comSEROTONIN, L-TRYPTOPHAN ARE PAT1 INHIBITORS

Figure 2. Electrophysiological analysis of mPAT1 function in X. laevis oocytes. A) Typical recording of inward current in an oocyte expressing mPAT1 at a holding potential of 60 mV in the presence of amino acids, biogenic amines, and derivatives (20 mM). B,C) Steady-state I-V relationships were measured by the two-electrode voltage clamp technique in the potential range 140 mV to 60 mV in oocytes expressing mPAT1 superfused with modied Barth solution at pH 6.5 B) in the presence of 20 mM l-tryptophan or of 5 mM glycine in the absence or presence of increasing concentrations (220 mM) of l-tryptophan, or C) in the presence of serotonin, tryptamine, and l-leucine at a concentration of 20 mM. Substrate-dependent currents were obtained as the difference measured in the absence and the presence of substrate.
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gesting a competitive mode of action of l-tryptophan. Similarly, serotonin, tryptamine, and 5-hydroxy-l-tryptophan also strongly reduced glycine-mediated inward directed current (Fig. 2C, Table 1) whereas l-leucine and d-tryptophan (20 mM) failed to inhibit currents evoked by glycine (Fig. 2C, Table 1). l-Lysine did not generate any signicant current itself, but its application resulted in a moderate inhibition of the glycine current (Table 1). Taken together, we conclude that serotonin, tryptamine, and l-tryptophan are recognized by the H amino acid symporter PAT1 expressed in different tissues from brain to intestine. They interact with the substrate binding site with an afnity similar to or even higher than the prototype substrates. They are, however, not transported by the PAT1 across the cell membrane. Since nothing is known about the substrate binding domain of PAT1, one could speculate that binding of these compounds restricts the conformational changes of the protein necessary for a complete substrate translocation cycle. We are not aware of any other example for a proteinogenic amino acid being able to inhibit the transport of another amino acids when not sharing the same transport pathway. With the predominant expression of PAT1 in the apical membrane in enterocytes, l-tryptophan ingested or released during protein breakdown in the gut could serve as the carriers natural inhibitor and determine the overall transport activity of PAT1. Even without knowing the concentration gradients after food intake and the ratio of free and peptide-bound l-tryptophan in the gut lumen or in the vicinity of the membrane and the substrate binding sites of PEPT1 and PAT1, at a recommended daily dietary allowance of 0.6 to 1 g for an adult, a concentration of free luminal l-tryptophan in the lower millimolar range (15 mM) can be anticipated, and this would be sufcient to cause PAT1 inhibition as shown here in vitro. Tryptamine originating from the decarboxylation of l-tryptophan by numerous bacterial species in the gut could also cause an inhibition of PAT1 activity. Moreover, 5-hydroxy-ltryptophan has been used clinically for decades to increase serotonin production in the brain (20). At oral doses of 300 mg per day (range: 50 to 3000 mg/day in different studies; for a review; see ref 20), a gut luminal concentration of 1 mM is conceivable, and this concentration corresponds well with its afnity constant for PAT1 inhibition. Since PAT1-mRNA is found not only in intestine but also in brain, liver, lung, placenta, and testis, our nding might also be relevant for its function in these tissues. PAT1 is, although with lower density found in neurons, mainly in lysosomal membranes and with much lower levels in the plasma membrane. Its inhibition by serotonin could be important for the transport for example of GABA or glycine across the cell membrane or for the efux of lysosomal proteolysis products from the organelle lumen to the cytosol (2). However, to dene the physiological role of PAT1 in brain and
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consequently that of its inhibition by l-tryptophan and derivatives needs further study.
This work was supported by Land Sachsen-Anhalt grant 3505A/0403L and by grants Bo 1857/1 and BR 1317/4 of the Deutsche Forschungsgemeinschaft. The expert technical assistance of Rainer Reichlmeir is gratefully acknowledged. This work will be part of the doctoral thesis of Linda Metzner.

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tion of high afnity inhibitors of the H /peptide transporter PEPT2. J. Biol. Chem. 277, 72877292 19. Takanaga, H., Mackenzie, B., Suzuki, Y., and Hediger, M. A. (2005) Identication of a mammalian proline transporter (SIT1, SLC6A20) with characteristics of classical system IMINO. J. Biol. Chem. 280, 8974 8984 20. Birdsall, T. C. (1998) 5-Hydroxytryptophan: a clinically effective serotonin precursor. Altern. Med. Rev. 3, 271280 Received for publication March 2, 2005. Accepted for publication April 27, 2005.

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