Mammalian Amino Acid Transporters Research Articles

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.

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.

News from the dark side!

News from the dark side!

Opposite Movements of the External Gate in Glutamate Transporters upon Binding Different Cotransported Ligands Measured by EPR

Hairpin two (HP2) has been proposed as the extracellular gate of glutamate transporters. To test this hypothesis, we use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter GltPh to examine conformational changes in HP2. Surprisingly, the two co-ligands Na+ and aspartate induce opposite movements of HP2. We find that Na+ binding to the apo state of the transporter opens the extracellular gate, while the subsequent binding of aspartate closes the gate. In addition, using voltage clamp fluorometry on the mammalian excitatory amino acid transporter EAAT3, we confirm that the opposite conformational changes of HP2 induced by Na+ and amino acid substrates also occur in mammalian amino acid transporters. Our findings are consistent with HP2 comprising the extracellular gate of glutamate transporters, and that Na+ binding opens and stabilizes the extracellular gate thereby allowing for amino acid substrate binding.

Ruminal and abomasal starch hydrolysate infusions selectively decrease the expression of cationic amino acid transporter mRNA by small intestinal epithelia of forage-fed beef steers

Although cationic amino acids (CAA) are considered essential to maximize optimal growth of cattle, transporters responsible for CAA absorption by bovine small intestinal epithelia have not been described. This study was conducted to test 2 hypotheses: 1) the duodenal, jejunal, and ileal epithelia of beef cattle differentially express 7 mRNA associated with 4 mammalian amino acid (AA) transport activities: y+ (CAT1), B0,+ (ATB0,+), b0,+ (b0,+AT and rBAT), and y+L (y+LAT1, y+LAT2, and 4F2hc), and 2) the expression of these mRNA is responsive to small intestinal luminal supply of AA substrates (derived from ruminal microbes) or glucose-derived energy (from starch hydrolysate, SH), or both. Eighteen ruminally and abomasally catheterized Angus steers (body weight = 260±17kg) fed an alfalfa cube-based diet at 1.33×net energy for maintenance requirement were assigned to 3 treatments (n=6): ruminal and abomasal water infusion (control); ruminal SH and abomasal water infusion; and ruminal water and abomasal SH infusion. The dosage of SH infusion amounted to 20% of metabolizable energy intake. After 14 or 16 d of infusion, steers were slaughtered, duodenal, jejunal, and ileal epithelia were harvested, and total RNA was extracted. The relative amounts of mRNA expressed by epithelia were quantified using real-time reverse transcription-PCR. All 7 mRNA species were expressed by the epithelium from each region, but their abundance differed among the regions. Specifically, duodenal expression of CAT1 and ATB0,+ mRNA was greater than jejunal or ileal expression; ileal expression of b0,+AT, rBAT, and y+LAT1 mRNA was greater than jejunal or duodenal expression, whereas the expression of y+LAT2 and 4F2hc mRNA did not differ among the 3 epithelia. With regard to SH infusion effect, ruminal infusion down-regulated or tended to down-regulate the jejunal expression of CAT1, rBAT, y+LAT2, and 4F2hc mRNA. Abomasal infusion down-regulated the jejunal expression of y+LAT2 mRNA and tended to down-regulate the jejunal expression of 4F2hc mRNA. This study characterized the pattern of CAA transporter mRNA expressed by growing beef cattle fed an alfalfa-based diet. Moreover, this study demonstrated that increasing the luminal supply of microbe-derived AA (by ruminal supplementation of SH) results in a reduced capacity of apical and basolateral membrane to transport of CAA, whereas increasing luminal glucose supply (by abomasal supplementation of SH) reduces only the basolateral transport capacity, assuming that CAA transporter mRNA content represents functional capacity.

Structure and Function of Cationic Amino Acid Transporters (CATs)

The CAT proteins (CAT for cationic amino acid transporter) are amongst the first mammalian amino acid transporters identified on the molecular level and seem to be the major entry path for cationic amino acids in most cells. However, CAT proteins mediate also efflux of their substrates and thus may also deplete cells from cationic amino acids under certain circumstances. The CAT proteins form a subfamily of the solute carrier family 7 (SLC7) that consists of four confirmed transport proteins for cationic amino acids: CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3). SLC7A4 and SLC7A14 are two related proteins with yet unknown function. One focus of this review lies on structural and functional differences between the different CAT isoforms. The expression of the CAT proteins is highly regulated on the level of transcription, mRNA stability, translation and subcellular localization. Recent advances toward a better understanding of these mechanisms provide a second focus of this review.

Differential Regulation of Amino Acid Transporter SNAT3 by Insulin in Hepatocytes

The liver is a metabolism and transfer center of amino acids as well as the prime target organ of insulin. In this report, we characterized the regulation of system N/A transporter 3 (SNAT3) in the liver of dietary-restricted mice and in hepatocytes treated with serum starvation and insulin. The expression of SNAT3 was up-regulated in dietary-restricted mice. The expression of SNAT3 protein was detected on the plasma membrane of hepatocyte-like H2.35 cells with a half-life of 6-8 h. When H2.35 cells were depleted of serum, the expression of SNAT3 was increased. An increased concentration of insulin, however, suppressed SNAT3 expression. Interestingly, the down-regulation of SNAT3 expression by insulin was blocked by the specific phosphoinositide 3-kinase inhibitor LY294002 and mammalian target of rapamycin inhibitor, but not by MAPK inhibitor PD98059, suggesting that insulin exerts its effect on SNAT3 through phosphoinositide 3-kinase-mammalian target of rapamycin signaling. Surface biotinylation assay showed an increased level of SNAT3 on the cell surface after 0.5 h of insulin treatment, although no effect was observed after 24 h of treatment. Consistently, the transport of the substrate l-histidine was increased with short, but not long, treatment by insulin in both H2.35- and SNAT3-transfected COS-7 cells. The L-histidine uptake was inhibited significantly by L-histidine followed by 2-endoamino-bicycloheptane-2-carboxylic acid and L-cysteine and to a lesser extent by L-alanine and aminoisobutyric acid, but was not inhibited by alpha-(methylamino)isobutyric acid, implying that uptake of L-histidine in H2.35 cells is primarily mediated by system N transporters. In conclusion, differential regulation of SNAT3 by insulin and serum starvation reinforces the functional significance of this transporter in liver physiology.

Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

The proteins that mediate polyamine translocation into eukaryotic cells have not been identified at the molecular level. To define the polyamine transport pathways in eukaryotic cells we have cloned a gene, LmPOT1, that encodes a polyamine transporter from the protozoan pathogen, Leishmania major. Sequence analysis of LmPOT1 predicted an unusual 803-residue polytopic protein with 9-12 transmembrane domains. Expression of LmPOT1 cRNA in Xenopus laevis oocytes revealed LmPOT1 to be a high affinity transporter for both putrescine and spermidine, whereas expression of LmPOT1 in Trypanosoma brucei stimulated putrescine uptake that was sensitive to inhibition by pentamidine and proton ionophores. Immunoblot analysis established that LmPOT1 was expressed predominantly in the insect vector form of L. major, and immunofluorescence demonstrated that LmPOT1 was localized predominantly to the parasite plasma membrane. To our knowledge this is the first molecular identification and characterization of a cell surface polyamine transporter in eukaryotic cells.

Aromatic Amino Acid Transporter AAT-9 of Caenorhabditis elegans Localizes to Neurons and Muscle Cells

The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport.

Lysinuric protein intolerance: mechanisms of pathophysiology

Heteromeric amino acid transporters (HATs) are composed of two subunits, a polytopic membrane protein (the light subunit) and a disulfide-linked type II membrane glycoprotein (the heavy subunit). HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y +L isoforms, asc, x c −, and b 0,+). The light subunits confer the amino acid transport specificity to the HAT. Two transporters of this family are relevant for inherited aminoacidurias. Mutations in any of the two genes coding for the subunits of system b 0,+ (rBAT and b 0,+AT) lead to cystinuria (MIM 220100). Transport defects in a system y +L isoform, composed of 4F2hc and y +LAT-1, result in lysinuric protein intolerance (LPI) (MIM 222700). In this case, only mutations in the light subunit y +LAT-1, but not in the heavy chain 4F2hc, cause the disease. LPI, in addition to affecting intestinal and renal reabsorption of amino acids, is a multisystemic disease affecting the urea cycle and presents also with symptoms related to the immune system. The pathogenesis of these alterations is less well, or not understood at all.

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

Localization and Role of Nitric Oxide Synthase and Endogenous Nitric Oxide Synthase Inhibitors in the Rabbit Lower Urinary Tract

We investigated the possible role of endogenous methylarginine derivatives, such as NG-monomethyl-L-arginine (L-NMMA), asymmetrical NG, NG-dimethyl-L-arginine (ADMA) and symmetrical NG, N'G-dimethyl-L-arginine (SDMA), for regulating nitric oxide synthase in the rabbit lower urinary tract. Strips of detrusor, trigone and proximal urethra were processed for the determination of endogenous methylarginines and L-arginine by automated high performance liquid chromatography. We also compared nitric oxide synthase activity and nitric oxide mediated functional responses to electrical field stimulation in the 3 regions. Nitric oxide synthase activity was measured by determining the conversion of [3H] L-arginine to [3H] L-citrulline. L-NMMA, ADMA and SDMA were detectable by high performance liquid chromatography in the detrusor, trigone and proximal urethra. Electrical field stimulation induced nitric oxide mediated prominent relaxation in the trigone and proximal urethra, while no relaxation response was observed in the detrusor. Exogenously applied 1 to 100 microM. L-NMMA and 1 to 100 microM. ADMA but not 100 microM. SDMA dependently inhibited Ca2+ dependent nitric oxide synthase activity in all regions, and electrical field stimulation induced relaxation in the trigone and proximal urethra. Inhibition with L-NMMA and ADMA was blocked in the presence of 3 mM. L-arginine but not by 3 mM. D-arginine. Three methylarginines and nitric oxide synthase are localized throughout the rabbit lower urinary tract. Endogenous L-NMMA and ADMA may be involved in regulating nitric oxide biosynthesis of the micturition reflex in the normal or disease state.

Heteromeric amino acid transporters: biochemistry, genetics, and physiology.

The heteromeric amino acid transporters (HATs) are composed of two polypeptides: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992, and the LSHATs have been described in the last three years. HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, x(c)(-), and b(0,+)). Members of the HAT family are the molecular bases of inherited primary aminoacidurias cystinuria and lysinuric protein intolerance. In addition to the role in amino acid transport, one HSHAT [the heavy subunit of the cell-surface antigen 4F2 (also named CD98)] is involved in other cell functions that might be related to integrin activation. This review covers the biochemistry, human genetics, and cell physiology of HATs, including the multifunctional character of CD98.

Amino acid transporters in the human placenta.

Amino acid transport across the human placenta is active, mediated by specific transporters in syncytiotrophoblast plasma membranes. Using functional criteria such as substrate specificity and sodium dependence, approximately 15 transport systems have been identified in the human placenta. Recently, the area of molecular biology of amino acid transporters has evolved rapidly and at least 25 cDNA clones coding for mammalian amino acid transporters or transporter subunits have been identified. The primary objective of this review is to integrate the available functional data on placental amino acid transport systems with recent molecular information on mammalian amino acid transporters. Furthermore, models for the mechanisms for net materno-fetal transfer of amino acids are discussed. Finally, the evidence to suggest that alterations in placental amino acid transport systems may play a crucial role in the regulation of fetal growth are presented briefly.

Heteromeric amino acid transporters explain inherited aminoacidurias

In the past 5 years, the first genes responsible for aminoacidurias caused by defects in renal reabsorption transport mechanisms have been identified. These diseases are type I and non-type I cystinuria and lysinuric protein intolerance. This knowledge came from the molecular characterization of the first heteromeric amino acid transporters in mammals. In 1992, rBAT and 4F2hc (genes SLC3A1 and SLC3A2, respectively, in the nomenclature of the Human Genome Organization) were identified as putative heavy subunits of mammalian amino acid transporters. In 1994, it was demonstrated that mutations in SLC3A1 cause type I cystinuria. Very recently, several light subunits of the heteromeric amino acid transporters have been identified. In 1999, a putative light subunit of rBAT (the SLC7A9 gene; complementary DNA and protein termed amino acid transporter) and a light subunit of 4F2hc (the SLC7A7 gene; cDNA and protein termed y+LAT-1) were shown to be the non-type I cystinuria and lysinuric protein intolerance genes, respectively. In this review, the characteristics of these heteromeric amino acid transporters and their role in these inherited aminoacidurias is described.

Identification and Characterization of a Na+-independent Neutral Amino Acid Transporter That Associates with the 4F2 Heavy Chain and Exhibits Substrate Selectivity for Small Neutral d- and l-Amino Acids

A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc.

Mammalian amino acid transport system y+ revisited: specificity and cation dependence of the interaction with neutral amino acids.

A reevaluation of the specificity of system y+, the classical transporter for cationic amino acids is presented. System y+ has been defined as a transporter for cationic amino acids that binds neutral amino acids with lower affinity in the presence of Na+. The discovery of other transporters for cationic amino has suggested that some properties, originally attributed to system y+, may relate to other transport systems. Uncertainty concerns mainly, the affinity for neutral amino acids and the cation dependence of this interaction. Neutral amino acids (13 analogues tested) were found to bind to system y+ in human erythrocytes with very low affinity. Inhibition constants (Kiy, mm) ranged between 14.2 mm and >400 mm, and the strength of interaction was similar in the presence of Na+, K+ or Li+ (145 mm). In choline medium, no interaction was detected up to 20 mm of the neutral amino acid. Guanidinium ion (5 mm, osmolarity maintained with choline) potentiated neutral amino acid binding; the effect was most important in the case of l-norvaline which aligned with guanidinium ion is equivalent to arginine. This suggests cooperative interaction at the substrate site. The specificity of system y+ was shown to be clearly distinct from that of system y+L, a cationic amino acid transporter that accepts neutral amino acids with high affinity in the presence of Na+ and which influenced the classical definition of system y+.

Molecular biology of mammalian plasma membrane amino acid transporters.

Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x(c)- and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

Expression cloning of a mammalian amino acid transporter or modifier by complementation of a yeast transport mutant.

A cDNA clone named L21 was isolated from L6 rat muscle cells by complementation of a yeast proline transport mutant. L21 cDNA has 2,268 bp and codes for a peptide of 228 amino acids with four potential transmembrane domains. The amino acid sequence of L21 shows no homology to any known proteins. Expression of L21 cDNA enables the mutant yeast to grow in proline as the sole nitrogen source and to transport proline, alpha-aminoisobutyric acid (AIB) and leucine. The Km for proline is about 1.0 mM. The substrate specificity of L21 expressed in yeast shows no striking similarity to known mammalian amino acid transporters.