Coupled Amino Acid Transporter Research Articles

SLC6A14, a Na+/Cl--coupled amino acid transporter, functions as a tumor promoter in colon and is a target for Wnt signaling.

SLC6A14 is a Na+/Cl−-coupled transporter for neutral and cationic amino acids. It is expressed at basal levels in the normal colon but is up-regulated in colon cancer. However, the relevance of this up-regulation to cancer progression and the mechanisms involved in the up-regulation remain unknown. Here, we show that SLC6A14 is essential for colon cancer and that its up-regulation involves, at least partly, Wnt signaling. The up-regulation of the transporter is evident in most human colon cancer cell lines and also in a majority of patient-derived xenografts. These findings are supported by publicly available TCGA (The Cancer Genome Atlas) database. Treatment of colon cancer cells with α-methyltryptophan (α-MT), a blocker of SLC6A14, induces amino acid deprivation, decreases mTOR activity, increases autophagy, promotes apoptosis, and suppresses cell proliferation and invasion. In xenograft and syngeneic mouse tumor models, silencing of SLC6A14 by shRNA or blocking its function by α-MT reduces tumor growth. Similarly, the deletion of Slc6a14 in mice protects against colon cancer in two different experimental models (inflammation-associated colon cancer and genetically driven colon cancer). In colon cancer cells, expression of the transporter is reduced by Wnt antagonist or by silencing of β-catenin whereas Wnt agonist or overexpression of β-catenin shows the opposite effect. Finally, SLC6A14 as a target for β-catenin is confirmed by chromatin immunoprecipitation. These studies demonstrate that SLC6A14 plays a critical role in the promotion of colon cancer and that its up-regulation in cancer involves Wnt signaling. These findings identify SLC6A14 as a promising drug target for the treatment of colon cancer.

Structural and functional studies on a prokaryotic homologue of the mammalian SLC7 cationic amino acid transporters

The Amino acid/Polyamine/Cationic transporter superfamily is among the largest of the sec-ondary active transporters. APC family members use either a proton or sodium gradient to drive substrate translocation across the membrane. Members of the APC superfamily are responsible for the uptake of amino acids and are found in all kingdoms of life. Amino acids, and their derivatives, are crucial for both prokaryotic and eukaryotic cell biology, being the building blocks of a variety of macromolecules and play important roles in metabolism and cell signalling. Therefore, the systems responsible for their transport are important targets of further study, in particular to gain greater insight into how these proteins recognise and discriminate between different types of amino acids. The transport mechanism of several APC members has been elucidated using a variety of structural, biophysical and functional approaches. However, these studies have focussed on sodium coupled transporters due to their function as neurotransmitter transporters. Here, the crystal structure of the first pro-ton-coupled amino acid transporter of the APC superfamily, from Geobacillus kaustophilus, GkApcT, is presented. GkApcT closely related to the mammalian cationic amino acid trans-porters (CATs) of the SLC7 family, responsible for the transport arginine and lysine into the cell. The mechanism of transport was further investigated using liposome-based functional assays to characterise the substrate specificity and kinetics. The crystal structure was used to guide a detailed site-directed mutagenesis study, which identified several conserved res-idues involved in substrate binding and proton-coupling. The current structure serves as a template to further understand proton coupled amino acid transport in bacteria and to un-derstand mammalian amino acid transport via the SLC7 and SLC36 families.

The D-amino acid transport by the invertebrate SLC6 transporters KAAT1 and CAATCH1 from Manduca sexta.

The ability of the SLC6 family members, the insect neutral amino acid cotransporter KAAT1(K+‐coupled amino acid transporter 1) and its homologous CAATCH1(cation anion activated amino acid transporter/channel), to transport D‐amino acids has been investigated through heterologous expression in Xenopus laevis oocytes and electrophysiological techniques. In the presence of D‐isomers of leucine, serine, and proline, the ms KAAT1 generates inward, transport‐associated, currents with variable relative potencies, depending on the driving ion Na+ or K+. Higher concentrations of D‐leucine (≥1 mmol/L) give rise to an anomalous response that suggests the existence of a second binding site with inhibitory action on the transport process. ms CAATCH1 is also able to transport the D‐amino acids tested, including D‐leucine, whereas L‐leucine acts as a blocker. A similar behavior is exhibited by the KAAT1 mutant S308T, confirming the relevance of the residue in this position in L‐leucine binding and the different interaction of D‐leucine with residues involved in transport mechanism. D‐leucine and D‐serine on various vertebrate orthologs B0 AT1 (SLC6A19) elicited only a very small current and singular behavior was not observed, indicating that it is specific of the insect neutral amino acid transporters. These findings highlight the relevance of D‐amino acid absorption in the insect nutrition and metabolism and may provide new evidences in the molecular transport mechanism of SLC6 family.

The anti-epileptic drug substance vigabatrin inhibits taurine transport in intestinal and renal cell culture models

The GABA-mimetic anti-epileptic drug substance vigabatrin is used against infantile spasms. In vitro and in vivo experiments have shown that vigabatrin is transported via the proton coupled amino acid transporter (PAT1) mediating at least parts of the intestinal absorption of the drug. However, such evidence does not preclude the involvement of other transporters. The aim of the present study was, therefore, to investigate if vigabatrin interacts with taurine transport. The uptake of taurine was measured in intestinal human Caco-2 and canine MDCK cell monolayers in the absence or presence of amino acids such as GABA and vigabatrin. Vigabatrin inhibits the uptake of taurine in Caco-2 and MDCK cells to 34±3 and 53±2%, respectively, at a concentration of 30mM. In Caco-2 cells the uptake of vigabatrin under neutral pH conditions is concentration-dependent and saturable with a Km-value of 27mM (logKm is 1.43±0.09). In conclusion, the present study shows that vigabatrin was able to inhibit the uptake of taurine in intestinal and renal cell culture models. Furthermore, uptake of vigabatrin in Caco-2 cells under neutral pH conditions was concentration-dependent and saturable and suggesting that vigabatrin partly was transported via a taurine transporter, which is likely to be TauT.

Intestinal absorption of the antiepileptic drug substance vigabatrin in Göttingen mini-pigs is unaffected by co-administration of amino acids

The anti-epileptic drug substance vigabatrin is used against infantile spasms. In vitro evidence suggests that vigabatrin is transported via the proton coupled amino acid transporter (PAT1). The aim of the present study was to investigate whether the intestinal absorption of vigabatrin in vivo was mediated via PAT1 in non-rodents. This was investigated by oral co-administration of vigabatrin and PAT1-ligands to Göttingen mini-pigs. Vigabatrin had an oral absorption fraction (Fabs) of 75-80%, and the maximal plasma concentration (Cmax) was reached within 0.5-1.0 h (tmax). Co-administration of vigabatrin and amino acids generally did not significantly affect Fa, Tmax or Cmax. However, co-administration with sarcosine prolonged the time to reach Cmax. After co-administration with amino acids, vigabatrin absorption showed a slightly lowered onset. This may indicate an effect of amino acids on either the rate of gastric emptying or an effect directly on the absorption of vigabatrin, possibly via inhibition of PAT1 or another drug transporter. In conclusion, co-administration of PAT1-ligands together with vigabatrin did not significantly alter the pharmacokinetic profile of vigabatrin.

Transport of the areca nut alkaloid arecaidine by the human proton-coupled amino acid transporter 1 (hPAT1)

The pyridine alkaloid arecaidine is an ingredient of areca nut preparations. It is responsible for many physiological effects observed during areca nut chewing. However, the mechanism underlying its oral bioavailability has not yet been studied. We investigated whether the H⁺-coupled amino acid transporter 1 (PAT1, SLC36A1), which is expressed in the intestinal epithelium, accepts arecaidine, arecoline, isoguvacine and other derivatives as substrates. Inhibition of L-[³H]proline uptake by arecaidine and derivatives was determined in Caco-2 cells expressing hPAT1 constitutively and in HeLa cells transiently transfected with hPAT1-cDNA. Transmembrane transport of arecaidine and derivatives was measured electrophysiologically in Xenopus laevis oocytes. Arecaidine, guvacine and isoguvacine but not arecoline strongly inhibited the uptake of L-[³H]proline into Caco-2 cells. Kinetic analyses revealed the competitive manner of L-proline uptake inhibition by arecaidine. In HeLa cells transfected with hPAT1-cDNA an affinity constant of 3.8 mm was obtained for arecaidine. Electrophysiological measurements at hPAT1-expressing X. laevis oocytes demonstrated that arecaidine, guvacine and isoguvacine are transported by hPAT1 in an electrogenic manner. We conclude that hPAT1 transports arecaidine, guvacine and isoguvacine across the apical membrane of enterocytes and that hPAT1 might be responsible for the intestinal absorption of these drug candidates.

Downregulation of the Creatine Transporter SLC6A8 by JAK2

Janus-activated kinase-2 (JAK2) participates in the regulation of the Na⁺-coupled glucose transporter SGLT1 and the Na⁺-coupled amino acid transporter SLC6A19. Concentrative cellular creatine uptake is similarly accomplished by Na⁺-coupled transport. The carrier involved is SLC6A8 (CreaT). The present study thus explored whether JAK2 regulates the activity of SLC6A8. To this end, cRNA encoding SLC6A8 was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2. Electrogenic creatine transport was determined in those oocytes by dual-electrode voltage-clamp experiments. In oocytes injected with cRNA encoding SLC6A8 but not in oocytes injected with water or with cRNA encoding JAK2 alone, addition of 1 mM creatine to the extracellular bath generated an inward current (I (crea)). In SLC6A8 expressing oocytes I (crea) was significantly decreased by coexpression of JAK2 or (V617F)JAK2 but not by coexpression of (K882E)JAK2. According to kinetic analysis, coexpression of JAK2 decreased the maximal transport rate without significantly modifying the affinity of the carrier. In oocytes expressing SLC6A8 and (V617F)JAK2 I (crea) was gradually increased by the JAK2 inhibitor AG490 (40 μM). In SLC6A8 and JAK2 coexpressing oocytes the decline of I (crea) following disruption of carrier insertion with brefeldin A (5 μM) was similar in the absence and presence of JAK2. In conclusion, JAK2 is a novel regulator of the creatine transporter SLC6A8, which downregulates the carrier, presumably by interference with carrier protein insertion into the cell membrane.

Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1

K+-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAAT1 shows a stretch of three glycines (G85–G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na+, K+, Li+) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(II)(1,10-phenanthroline)3, showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na+ protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.

Rectal Absorption of Vigabatrin, a Substrate of the Proton Coupled Amino Acid Transporter (PAT1, Slc36a1), in Rats

To investigate the rectal absorption of vigabatrin in rats, based on the hypothesis that PAT1 (Slc36a1) is involved. Male Sprague-Dawley rats were dosed rectally with five different gels, varying in buffer capacity, the amount of vigabatrin, and co-administration of proline or tryptophan. Western blotting was used to detect rPAT1 in rat rectal epithelium. X. Laevis oocytes were injected with SLC36A1 cRNA for the expression of hPAT1, prior to two-electrode voltage clamp measurements. rPAT1 protein was present in rat rectal epithelium. Approximately 7%-9% of a 1mg/kg vigabatrin dose was absorbed after rectal administration, regardless of the formulation used. Increasing the dose of vigabatrin 10-fold decreased the absolute bioavailability to 4.2%. Co-administration of proline or tryptophan changed the pharmacokinetic profile, indicating a role of PAT1 in the rectal absorption of vigabatrin. Transport of vigabatrin via hPAT1 expressed in X. Laevis oocytes had a K(m) of 5.2 ± 0.6mM and was almost completely inhibited by tryptophan. Although vigabatrin is a PAT1 substrate and the rPAT1 protein is expressed in the rectum epithelium, vigabatrin has low rectal absorption in rats.

Stimulation of the amino acid transporter SLC6A19 by JAK2

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na+ coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, V617FJAK2 or inactive K882EJAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2mM) to the bath generated a current (Ile), which was significantly increased following coexpression of JAK2 or V617FJAK2, but not by coexpression of K882EJAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40μM) resulted in a gradual decline of Ile. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of Ile following inhibition of carrier insertion by brefeldin A (5μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.

Cellular localization and protein concentration of the SNAT2 transporter in rat mammary gland during pregnancy and lactation

The sodium coupled amino acid transporter SNAT2 is expressed in mammary gland (MG). There is a special amino acid demand for cellular differentiation during pregnancy and for protein milk secretion during lactation in the MG. The purpose of the present work was to assess the amount and localization of SNAT along pregnancy and lactation in rat MG. Western blot analysis in MG of virgin rats showed the presence of a single band with a MW of 60 kD corresponding to SNAT2 protein. The intensity of this band was maintained during pregnancy and decreased dramatically one day before delivery. During lactation, SNAT2 increased rapidly after 5 days and remains constant along lactation and decreased after post-weaning. Immunohistochemical analysis revealed that MG SNAT2 of virgin rats was exclusively located in adipose cells. During gestation, there was an increase in the staining of SNAT2 in epithelial cells that will synthesize milk and decrease before delivery. During lactation there was a significant increase in the amount of SNAT2 in epithelial cells. Electron immunomicroscopy analysis of SNAT2 in MG during peak of lactation indicated that the SNAT2 protein was retained in vesicles originated from the endoplasmic reticulum and then translocated to the basolateral membrane of the epithelial cells. Our results suggested that presence of SNAT2 in the different cells and stages depends on the needs of the MG. CONACYT grant No 46134

Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2)

The H+-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[3H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC50 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.

M1720 Amino Acid Sensing by Enteroendocrine STC-1 Cells: Role of the Na+-Coupled Neutral Amino Acid Transporter 2 (Snat2)

M1720 Amino Acid Sensing by Enteroendocrine STC-1 Cells: Role of the Na+-Coupled Neutral Amino Acid Transporter 2 (Snat2)

Abstract 4838: Alpha-methyltryptophan, a blocker of the amino acid transporter SLC6A14, induces autophagy and apoptosis in SLC6A14-positive breast cancer and colon cancer cell lines

Abstract SLC6A14 (also known as ATB0,+) is a Na+/Cl− -coupled amino acid transporter that transports 18 of the 20 proteinogenic amino acids, including all essential amino acids. Normal tissues express this transporter at low levels, whereas tumor tissues (colon, breast, and cervix) upregulate this transporter several-fold. The unique functional features of SLC6A14 (broad substrate specificity and multiple driving forces) make it an ideal transporter for tumor cells to maintain amino acid nutrition necessary for enhanced cell proliferation. We speculate that SLC6A14 could not only be an ideal tumor-specific delivery system for amino acid-based anti-cancer drugs but also be a direct drug target for cancer chemotherapy. Recently we identified alpha-methyltryptophan (ALT) as a blocker of SLC6A14. Here we examine the potential of this blocker as an anti-cancer agent in SLC6A14-positive breast and colon cancer cells. Treatment of MCF7 (a breast cancer cell line), HCT116 and LS174T (colon cancer cell lines) with ALT causes amino acid starvation as evidenced by the upregulation of asparagine synthetase and CHOP. These three cell lines are SLC6A14-positive. In contrast, ALT treatment of breast cancer cell line MDA-MB231, which is SLC6A14-negative, does not show any evidence of amino acid starvation. Similarly, ALT treatment has no effect on normal breast and colon epithelial cell lines (MCF10A and CCD841, respectively). These cell lines also do not express SLC6A14. These data show that ALT-induced amino acid starvation is mediated specifically through the blockade of SLC6A14. Since nutrient signaling is also associated with changes in mTOR pathway, we examined the influence of ALT treatment on phosphorylation of S6 and S6kinase. Blockade of SLC6A14 with ALT in SLC6A14-positive cancer cell lines leads to decreased mTOR signaling as evidenced by decreased phosphorylation of S6 and S6kinase. Again, such changes are not seen in SLC6A14-negative cancer or normal cell lines. The initial response of the SLC6A14-positive cancer cell lines to ALT treatment is induction of autophagy. ALT-induced autophagy is documented in these cells by light and electron microscopic examination as well as by biochemical means. Inhibition of autophagy with 3-methyladenine under these conditions leads to apoptosis. We conclude that SLC6A14 is a potential drug target for cancer chemotherapy and that ALT could serve as a lead compound for the future design of high-affinity blockers of SLC6A14. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4838.

Transport of the Photodynamic Therapy Agent 5-Aminolevulinic Acid by Distinct H+-Coupled Nutrient Carriers Coexpressed in the Small Intestine

5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.

The Orally Active Antihyperglycemic Drug β-Guanidinopropionic Acid Is Transported by the Human Proton-Coupled Amino Acid Transporter hPAT1

The orally administered creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases plasma glucose levels by increasing the sensitivity to insulin. This effect is based on a beta-GPA induced expression of mRNA and total protein content of the insulin-responsive glucose transporter GLUT4. Although the oral availability of beta-GPA is well established, the underlying uptake mechanism has not yet been studied. We investigated whether the H(+)-coupled amino acid transporter PAT1, which is expressed in the apical membrane of intestinal cells, accepts guanidine derivatives as substrates. Uptake of l-[(3)H]proline into Caco-2 cells expressing hPAT1 constitutively was strongly inhibited by beta-GPA and its derivatives guanidinoacetic acid (GAA) and 4-guanidinobutyric acid (4-GBA). Competition assays revealed apparent affinity constants of about 1.5 mM. Electrophysiological measurements at hPAT1-expressing Xenopus laevis oocytes unequivocally demonstrated that beta-GPA, GAA and 4-GBA are effectively transported by this transport system in an electrogenic manner. We conclude that hPAT1 might be responsible for the intestinal absorption of beta-GPA thereby allowing its oral administration. Moreover, with beta-GPA we identified a new high affinity hPAT1 substrate that might be an interesting starting point for future drug design-drug delivery strategies.

Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy

ATB(0,+) [SLC6A14 (solute carrier family 6 member 14)] is an Na(+)/Cl(-)-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB(0,+) in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB(0,+). The transport process is Na(+)/Cl(-)-dependent with an Na(+)/Cl(-)/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of alpha-methyltryptophan as a blocker, not a transportable substrate, for ATB(0,+). ATB(0,+) can transport 18 of the 20 proteinogenic amino acids. alpha-Methyltryptophan blocks the transport function of ATB(0,+) with an IC(50) value of approximately 250 muM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that alpha-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB(0,+) is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB(0,+)-positive tumour cells with alpha-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB(0,+)-negative cell lines are not affected. The blockade of ATB(0,+) in these cells with alpha-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB(0,+) as a drug target for cancer chemotherapy.

Akt Mediates the Effect of Insulin on Epithelial Sodium Channels by Inhibiting Nedd4-2

The epithelial sodium channel (ENaC) plays an important role in transepithelial Na(+) absorption; hence its function is essential for maintaining Na(+) and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1(K179M), decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser(342) and Ser(428), completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na(+) absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)

The H+-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly–Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly–Sar uptake through a reduction in capacity (Vmax) without any effect on affinity (Km). The reduction in dipeptide transport was dependent upon both extracellular Na+ and apical pH but was not observed in the presence of the selective Na+/H+ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H+-electrochemical gradient which, in turn, acts as the driving force for H+-coupled solute transport. Uptake of β-alanine, a substrate for the H+-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H+-electrochemical gradient and may, therefore, be a site for both nutrient–drug and drug–drug interactions in the small intestine.

Substrate specificity of the amino acid transporter PAT1

The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic alpha-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.