Coupled Amino Acid Transporter Research Articles

Indirect regulation of the intestinal H+‐coupled amino acid transporter hPAT1 (SLC36A1)

A H(+)-coupled amino acid transporter has been characterised functionally at the brush border membrane of the human intestinal cell line Caco-2. This carrier, hPAT1 (human Proton-coupled Amino acid Transporter 1) or SLC36A1, has been identified recently at the molecular level and hPAT1 protein is localised to the brush border membrane of human small intestine. hPAT1 transports both amino acids (e.g., beta-alanine) and therapeutic agents (e.g., D-cycloserine). In human Caco-2 cells, hPAT1 function (H(+)/amino acid symport) is associated with a decrease in intracellular pH (pH(i)), which selectively activates the Na(+)/H(+) exchanger NHE3, and thus maintains pH(i) and the driving force for hPAT1 function (the H(+) electrochemical gradient). This study provides the first evidence for regulation of hPAT1 function. Activation of the cAMP/protein kinase A pathway in Caco-2 cell monolayers either using pharmacological tools (forskolin, 8-br-cAMP, [(11,22,28)Ala]VIP) or physiological activators (the neuropeptides VIP and PACAP) inhibited hPAT1 function (beta-alanine uptake) at the apical membrane. Under conditions where NHE3 is inactive (the absence of Na(+), apical pH 5.5, the presence of the NHE3 inhibitor S1611) no regulation of beta-alanine uptake is observed. Forskolin and VIP inhibit pH(i) recovery (NHE3 function) from beta-alanine-induced intracellular acidification. Immunocytochemistry localises NHERF1 (NHE3 regulatory factor 1) to the apical portion of Caco-2 cells where it will interact with NHE3 and allow PKA-mediated phosphorylation of NHE3. In conclusion, we have shown that amino acid uptake via hPAT1 is inhibited by activators of the cAMP pathway indirectly through inhibition of NHE3 activity.

Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system

To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (alpha type) containing liver cDNA library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. Nineteen colonies were selected and sequenced. Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calreticulin, one was human serum albumin (ALB) gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase, three were Homo sapiens Na(+) and H(+) coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function. The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.

A novel physiological mechanism of glycine-induced immunomodulation: Na+-coupled amino acid transporter currents in cultured brain macrophages.

Glycine is known to modulate immune cell responses. However, the physiological mechanisms underlying inhibitory effects of glycine on macrophages are not well understood. Here we show that glycine is capable of inducing inward currents in brain macrophages (microglia). In contrast to glycine, the glycine receptor agonist taurine failed to elicit currents. Glycine-evoked currents of brain macrophages were unaffected by strychnine, Cl(-)-free extracellular solution, N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (NFPS) and amoxapine, but were abolished upon omission of extracellular Na(+). Furthermore, glycine caused increases in the intracellular Na(+) concentration and pronounced membrane depolarization. Glycine-evoked depolarization was Na(+) dependent and occurred independently of the intracellular Cl(-) concentration. Similarly to glycine, glutamine and alpha-(methylamino)isobutyric acid (MeAIB) elicited inward currents in brain macrophages. In the presence of either glutamine or MeAIB, glycine-induced currents were inhibited. It is concluded that neither functional glycine receptors nor glycine transporters are expressed in brain macrophages. We suggest that glycine mediates its effects by activation of system A Na(+)-coupled neutral amino acid transporters.

Transport of amino acid-based prodrugs by the Na+- and Cl(-) -coupled amino acid transporter ATB0,+ and expression of the transporter in tissues amenable for drug delivery.

We evaluated the potential of the Na(+)- and Cl(-)-coupled amino acid transporter ATB(0,+) as a delivery system for amino acid-based prodrugs. Immunofluorescence analysis indicated that ATB(0,+) is expressed abundantly on the luminal surface of cells lining the lumen of the large intestine and the airways of the lung and in various ocular tissues, including the conjunctival epithelium, the tissues easily amenable for drug delivery. We screened a variety of beta-carboxyl derivatives of aspartate and gamma-carboxyl derivatives of glutamate as potential substrates for this transporter using heterologous expression systems. In mammalian cells expressing the cloned ATB(0,+), several of the aspartate and glutamate derivatives inhibited glycine transport via ATB(0,+). Direct evidence for ATB(0,+)-mediated transport of these derivatives was obtained in Xenopus laevis oocytes using electrophysiological methods. Exposure of oocytes, which express ATB(0,+) heterologously, to aspartate beta-benzyl ester as a model derivative induced inward currents in a Na(+)- and Cl(-)-dependent manner with a Na(+)/Cl(-)/aspartate beta-benzyl ester stoichiometry of 2:1:1. ATB(0,+) transported not only the beta-carboxyl derivatives of aspartate and the gamma-carboxyl derivatives of glutamate but also valacyclovir, which is an alpha-carboxyl ester of acyclovir with valine. The transport of valacyclovir via ATB(0,+) was demonstrable in both heterologous expression systems. This process was dependent on Na(+) and Cl(-). The ability of ATB(0,+) to transport valacyclovir was comparable with that of the peptide transporter PEPT1. These findings suggest that ATB(0,+) has significant potential as a delivery system for amino acid-based drugs and prodrugs.

Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2.

The human orthologue of the H(+)-coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco-2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of alpha-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na(+)-independent, but was energized by an inwardly directed H(+) gradient. hPAT1 interacted with glycine, L-alanine, L-proline, alpha-aminoisobutyrate (AIB) and gamma-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the D-isomers of alanine and proline. With serine and cysteine, though the L-isomers did not interact with hPAT1 to any significant extent, the corresponding D-isomers were recognized as substrates. With proline and alanine, the affinity was similar for L- and D-isomers. However, with cysteine and serine, the D-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding L-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H(+)-coupled amino acid transport system in Caco-2 cells. Furthermore, there was a high degree of correlation (r(2) = 0.93) between the relative potencies of various amino acids to inhibit the H(+)-coupled MeAIB transport measured with native Caco-2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 cells. These data suggest that hPAT1 is responsible for the H(+)-coupled amino acid transport expressed in the apical membrane of Caco-2 cells.

Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+).

Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract.

Primary Structure, Genomic Organization, and Functional and Electrogenic Characteristics of Human System N 1, a Na+- and H+-coupled Glutamine Transporter

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.

Molecular mode of action of the antifungal beta-amino acid BAY 10-8888.

BAY 10-8888 is a cyclic beta-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H+-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a KT (Michaelis constant of the transport reaction) of 0.95 mM and a Vmax of 18.9 nmol x min-1 x 10(7) cells-1. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.

Cloning and characterization of a potassium-coupled amino acid transporter.

Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na+ or H+ gradients. The present work extends the Na+ and H+ dogma by including the H+ and K+ paradigm. Lepidopteran insect larvae have a high K+ and a low Na+ content, and their midgut cells lack Na+/K+ ATPase. Instead, an H+ translocating, vacuolar-type ATPase generates a voltage of approximately -240 mV across the apical plasma membrane of so-called goblet cells, which drives H+ back into the cells in exchange for K+, resulting in net K+ secretion into the lumen. The resulting inwardly directed K+ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K+-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes alpha-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K+, Na+, and to a lesser extent Li+ were accepted as cotransported ions, but K+ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl(-)-dependent, and the K+/Na+ selectivity increased with hyperpolarization of oocytes, reflecting the increased K+/Na+ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na+ and Cl(-)-coupled neurotransmitter transporter family.

Possible existence of Na +-coupled amino acid transport in the pigmented rabbit conjunctiva

The objective of the present study was to determine whether Na +-amino acid cotransport contributed to the short-circuit current (I sc) in the isolated pigmented rabbit conjunctiva. Glycine, L-arginine, D-arginine, and L-glutamic acid were the amino acids tested, and bioelectric measurements were made in the Using chamber. The I sc was increased from 4% (L-glutamic acid) to 44% (L-arginine). The EC 50 was 0.35 mM for glycine, 0.06 mM for L-arginine, 0.16 mM for D-arginine, and 1 mM for L-glutamic acid. No elevation in I sc was seen in the absence of Na + in the mucosal bathing fluid. The above findings are consistent with the possible existence of a Na +-amino acid cotransport process on the apical side of the pigmented rabbit conjunctiva.

D‐Cycloserine transport in human intestinal epithelial (Caco‐2) cells: mediation by a H + ‐coupled amino acid transporter

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.

Transport of 2-aminoisobutyric acid in cultured endothelial cells

Conflicting reports exist concerning the presence of a Na +-coupled amino acid transport system in cultured endothelial cells. We have employed a non-metabolizable analog, 2-aminoisobutyric acid (AIB), to investigate the activity of Na +-dependent amino acid transport in cultured human umbilical vein endothelial cells (HUVEC). We found a pronounced saturable, Na +-dependent component of AIB uptake in ‘fresh’ (non-starved) HUVEC. The Na +-dependent component accounted for 78% of total AIB uptake with a high sensitivity to external Na +. The accumulation of AIB was inhibited by ouabain preincubation, consistent with the energetics of Na +-coupled transport. Amiloride, an epithelial Na + channel blocker, also inhibited AIB transport at high concentration. The results strongly support the presence of a Na +-coupled transport system of amino acid in HUVEC.

Specific inhibition of the Na(+)-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus strains, was found to cause growth inhibition; therefore, the use of amiloride for the isolation of motility mutants was difficult. On the other hand, phenamil, an amiloride analog, inhibited motor rotation without affecting cell growth. A concentration of 50 microM phenamil completely inhibited the motility of strain RA-1 but showed no effect on the membrane potential, the intracellular pH, or Na(+)-coupled amino acid transport, which was consistent with the fact that there was no effect on cell growth. Kinetic analysis of the inhibition of motility by phenamil indicated that the inhibition was noncompetitive with Na+ in the medium. A motility mutant was isolated as a swarmer on a swarm agar plate containing 50 microM phenamil. The motility of the mutant showed an increased resistance to phenamil but normal sensitivity to amiloride. These results suggest that phenamil and amiloride interact at different sites on the motor. By examining various bacterial species, phenamil was found to be a specific and potent inhibitor for the Na(+)-driven flaggellar motors not only in various strains of alkalophilic Bacillus spp. but also in a marine Vibrio sp.

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K(m)-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V(max app) was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 x 10(6) vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H(+)-translocating enzyme activities revealed K(m app) values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Delta muH(+) while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.

Amiloride, a specific inhibitor for the Na+-driven flagellar motors of alkalophilic Bacillus.

Na+-driven flagellar motors of alkalophilic Bacillus were found to be inhibited by amiloride, a potent inhibitor for many Na+-coupled systems. A concentration of 0.5 mM of amiloride completely inhibited motility but showed almost no effect on the membrane potential, the intracellular pH homeostasis, and the ATP content of the cells. Furthermore, the activity of a Na+-coupled amino acid transport system was reduced only by half by this concentration of amiloride. Thus, the inhibition of motility of alkalophilic Bacillus by amiloride was rather specific. The inhibition of motility produced by amiloride was restored by increasing Na+ concentrations in the medium. Kinetic analysis of the data revealed that the inhibition was competitive with respect to the concentration of Na+ in the medium. Therefore, it is quite logical to assume that amiloride inhibits the rotation of the Na+-driven flagellar motors of alkalophilic Bacillus by competing with Na+ at the force-generating site of the motor. Some amiloride analogs known to selectively inhibit Na+ channels were potent inhibitors for the flagellar motors, suggesting that the Na+-interacting site of the motors has some similarity to that of the Na+ channels.

Amino acid transport in a kidney epithelial cell line (MDCK): Characteristics of Na +/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na +-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na +-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na +-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na +-stimulated MeAIB transport activity and Na +,K +,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na +-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na +-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na + dependence of amino acid transport in vesicles suggested a Na + symport mechanism with a 1:1 stoichiometry between Na + and amino acid.

Partial Purification of Amino Acid Transport Systems in Ehrlich Ascites Tumor Cell Plasma Membranes

Ehrlich ascites tumor cell plasma membranes were subjected to sequential selective protein extraction to identify protein components associated with amino acid transport. These membranes were extracted with Triton X-100 followed by 2,3-dimethylmaleic anhydride. Approximately 80% of the membrane proteins were extracted by these procedures while the original lipids were largely retained (approximately 70%). The quantity of carbohydrate per milligram protein in the residue increased on extraction, consistent with an enrichment of glycoprotein in the residue. The residual vesicles display the characteristic properties of Na+-coupled amino acid transport. These properties include Na+-stimulated uptake and Na+-gradient-stimulated uptake leading to an accumulation of the solute against its chemical gradient as well as inhibition of uptake by a competitive amino acid, L-methionine. The extracted vesicles exhibit a peak level of alpha-aminoisobutyrate uptake six times greater than that expected from equilibration of alpha-aminoisobutyrate. This accumulation is greater than that obtained with native vesicles, albeit slower. The accelerated exchange diffusion of L-leucine is not measurable in the residual vesicles after dimethylmaleic acid anhydride treatment, although it can be measured after Triton extraction. These results are consistent with the conclusion that the amino acid transport systems "A" (Na+-coupled) and "L" (Na+-independent) in Ehrlich cells, though having overlapping specificities for amino acids, and distinct physical entities.