Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A

We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.     

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.     

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.     

Evidence for the transport of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A

We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including α-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na⁺-coupled manner and the transport of cationic amino acids in a Na⁺-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y⁺L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.     

A novel system A isoform mediating Na+/neutral amino acid cotransport

A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Na(+)-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by alpha-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K(m)) of 200-500 microm. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to alpha-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.     

Amino acid transporter ATA2 is stored at the trans-Golgi network and released by insulin stimulus in adipocytes

Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of alpha-(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-d-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.     

Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2

We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of alpha-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface.     

Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A

Treatment of HepG2 cells with forskolin led to 60-100% stimulation of system A activity, measured as the Na+-dependent uptake of alpha-(methylamino)isobutyric acid. The stimulation was reproducible with cholera toxin and dibutyryl cAMP, and inhibitable by H7, a non-specific protein kinase inhibitor. The stimulatory effect was eliminated by cycloheximide and actinomycin D. The forskolin effect was associated with an increase in the maximal velocity of the transport system, with no change in substrate affinity. These cells express three different subtypes of system A (ATA1, ATA2, and ATA3). Treatment with forskolin increased the steady-state levels of ATA1 and ATA2 mRNAs, but decreased that of ATA3 mRNA.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A

We investigated the molecular mechanism involved in the adaptive regulation of the amino acid transport system A, a process in which amino acid starvation induces the transport activity. These studies were done with rat C6 glioma cells. System A activity in these cells is mediated exclusively by the system A subtype, amino acid transporter A2 (ATA2). The other two known system A subtypes, ATA1 and ATA3, are not expressed in these cells. Exposure of these cells to an amino acid-free medium induces system A activity. This process consists of an acute phase and a chronic phase. Laser-scanning confocal microscopic immunolocalization of ATA2 reveals that the acute phase is associated with recruitment of preformed ATA2 from an intracellular pool to the plasma membrane. In contrast, the chronic phase is associated with an induction of ata2 gene expression as evidenced from the increase in the steady-state levels of ATA2 mRNA, restoration of the intracellular pool of ATA2 protein, and blockade of the induction by cycloheximide and actinomycin D. The increase in system A activity induced by amino acid starvation is blocked specifically by system A substrates, including the non-metabolizable alpha-(methylamino)isobutyric acid.     

Structure,tissue expression pattern,and function of the amino acid transporter rat PAT2

The second member of the PAT (proton-coupled amino acid transporter) family of H(+)-coupled, pH-dependent, Na(+)-independent amino acid transporters was isolated from a rat lung cDNA library. The cDNA for rat PAT2 is 2396bp in length, including 70bp of 5'UTR and a poly(A) tail. The transporter gene, consisting of 10 exons, is located on rat chromosome 10q22. The cDNA codes for a protein of 481 amino acids with 72% identity (over 449 amino acids) with rat PAT1. Tissue expression studies demonstrate that mRNA abundance is generally low with highest levels being detected in lung and spleen, with lower levels in the brain, heart, kidney, and skeletal muscle. Functional expression in either mammalian cells or Xenopus laevis oocytes demonstrates that rat PAT2 mediates pH-dependent, Na(+)-independent uptake of glycine, proline, and alpha(methyl)aminoisobutyric acid (MeAIB). In conclusion PAT2 has a limited tissue distribution, higher affinity (Michaelis-Menten constant for glycine uptake between 0.49 and 0.69mM), and distinct substrate specificity compared to PAT1.     

The C-terminal domain of the neutral amino acid transporter SNAT2 regulates transport activity through voltage-dependent processes

SNAT (sodium-coupled neutral amino acid transporter) 2 belongs to the SLC38 (solute carrier 38) family of solute transporters. Transport of one amino acid molecule into the cell is driven by the co-transport of one Na(+) ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. In the present paper, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of this deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (<0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were normal in the truncated transporter. SNAT2(Del C-ter) (13 residues deleted from the C-terminus) showed 3-fold higher apparent affinity for alanine, and 2-fold higher Na(+) affinity compared with wild-type SNAT2, suggesting that the C-terminus is not required for high-affinity substrate and Na(+) interaction with SNAT2. The pH sensitivity of amino acid transport was retained partially after the truncation. In contrast with the truncation after TM (transmembrane domain) 11, the deletion of TM11 resulted in an inactive transporter, most probably due to a defect in cell surface expression. Taken together, the results demonstrate that the C-terminal domain of SNAT2 is an important voltage regulator that is required for a normal amino acid translocation process at physiological membrane potentials. However, the C-terminus appears not to be involved in the regulation of membrane expression.     

Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies on amino acid transport in murine P388 leukemia cells in vitro. Presence of system y+

The transport of glycine and L-lysine into murine P388 leukemia cells has been examined. Glycine transport appears to be shared by both systems A and ASC in P388 cells. Glycine transport is Na+-dependent and is effectively blocked by alpha-(methylamino)isobutyric acid, threonine and alanine but only a marginal reduction in transport is seen with 100-fold excess cold 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. System gly is not expressed in P388 cells. Lysine is largely transported by a Na+-independent, pH-insensitive system with a Km of 0.079 mM. Lysine transport is relatively unaffected by the addition of 100-fold excess cold alpha-(methylamino)isobutyric acid, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and the anionic amino acids, L-glutamate and L-aspartate. A partial inhibition of lysine transport was observed with L-threonine and L-leucine while L-arginine and L-histidine radically decreased lysine transport. Lysine appears to be transported by a system similar to the system y+ seen in cultured human fibroblasts, Ehrlich ascites cells, and hepatoma cell lines.     

Characterisation and cloning of a Na(+)-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B(0) transporter family

Na(+)-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B(0). This transporter is shown to differ in specificity from the B(0) transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B(0) transporter family we have isolated a cDNA encoding the NBL-1 cell System B(0) transporter. When expressed in Xenopus oocytes the clone catalysed Na(+)-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na(+)-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B(0)/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B(0) transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.     

The sodium-coupled neutral amino acid transporter SNAT2 mediates an anion leak conductance that is differentially inhibited by transported substrates

The sodium-coupled neutral amino acid transporter SNAT2 mediates cellular uptake of glutamine and other small, neutral amino acids. Here, we report the existence of a leak anion pathway associated with SNAT2. The leak anion conductance was increased by, but did not require the presence of, extracellular sodium. The transported substrates L-alanine, L-glutamine, and alpha-(methylamino)isobutyrate inhibited the anion leak conductance, each with different potency. A transporter with the mutation H-304A did not catalyze alanine transport but still catalyzed anion leak current, demonstrating that substrate transport is not required for anion current inhibition. Both the substrate and Na+ were able to bind to the SNAT2H-304A transporter normally. The selectivity sequence of the SNAT2H-304A anion conductance was SCN->NO3->I->Br->Cl->Mes-. Anion flux mediated by the more hydrophobic anion SCN- was not saturable, whereas nitrate flux demonstrated saturation kinetics with an apparent Km of 29 mM. SNAT2, which belongs to the SLC38 family of transporters, has to be added to the growing number of secondary, Na+-coupled transporters catalyzing substrate-gated or leak anion conductances. Therefore, we can speculate that such anion-conducting pathways are general features of Na+-transporting systems.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

Cloning and functional characterization of a new subtype of the amino acid transport system N

We have cloned a new subtype of theamino acid transport system N2 (SN2 or second subtype of system N) fromrat brain. Rat SN2 consists of 471 amino acids and belongs to therecently identified glutamine transporter gene family that consists ofsystem N and system A. Rat SN2 exhibits 63% identity with rat SN1. Italso shows considerable sequence identity (50-56%) with themembers of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung,stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediatesNa⁺-dependent transport of several neutral amino acids,including glycine, asparagine, alanine, serine, glutamine, andhistidine. The transport process is electrogenic, Li⁺tolerant, and pH sensitive. The transport mechanism involves the influxof Na⁺ and amino acids coupled to the efflux ofH⁺, resulting in intracellular alkalization. Proline,-(methylamino)isobutyric acid, and anionic and cationic amino acidsare not recognized by rat SN2.

     

Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition

The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.