Basis for substrate preference of amino acid transport system L over amino acid transport system A.
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Amino Acids↗
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Molecular cloning of mouse amino acid transport system B0, a neutral amino acid transporter related to Hartnup disorder.
Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B(0). Here we have characterized a novel member of the Na(+)-dependent neurotransmitter transporter family (B(0)AT1) isolated from mouse kidney, which shows all properties of system B(0). Flux experiments showed that the transporter is Na(+)-dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB(0)AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B(0)AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B(0) transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB(0)AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder.
Amino Acid Sequence↗
Regulation of amino acid transport systems by amino acid depletion and supplementation in monolayer cultures of rat hepatocytes.
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Aminoisobutyric Acids↗
Endogenous amino acid transport systems and expression of mammalian amino acid transport proteins in Xenopus oocytes.
Oocyte amino acid transport has physiological significance to oocytes and practical importance to molecular biologists and transport physiologists. Expression of heterologous mRNA in Xenopus oocytes is currently being used to help clone cDNAs for amino acid transporters and their effectors. A major question to be resolved in many of these studies is whether the injected mRNA codes for a transporter or an activator of an endogenous system. Nevertheless, the cDNAs of several families of amino acid transporters or their activators appear already to have been cloned. One such transporter is the anion exchanger, band 3, which may also transport glycine and taurine under some important physiological conditions such as hypoosmotic stress. Site-directed mutagenesis of band 3 has already shown that an amino acid residue believed to be at or near the active site nevertheless does not appear to influence Cl- transport in Xenopus oocytes expressing the modified band 3 protein. Continuation of such studies along with examination of transport of all possible substrates of band 3 should yield insight into the relationship between the structure and function of this transporter. Each of three other families not only contains amino acid transporters, but also appears to contain members that serve as transporters of neurotransmitters or their metabolites. Because of the distinct structural differences in the preferred substrates of different transporters within some of these families, elucidation of the tertiary and possibly quaternary structural relationships among the members of such families may reveal transport mechanisms. In addition, the grouping of neurotransmitters or their metabolites according to the family to which their transport systems and transporters belong could yield insight into mechanisms of brain development, function and evolution. Another family of transporters for cationic amino acids also serves, at least in one case, as a viral receptor. Hence, these or other transporters also could conceivably function in eggs as receptors for sperm and, more broadly, in cell-cell interactions as well as in amino acid transport. Moreover, a family of apparent amino acid transport activators are homologous to a family of glycosidases, so these activators could also serve to recognize carbohydrate structures on other cells or the extracellular matrix. Some of these activators appear to increase more than one amino acid transport activity in Xenopus oocytes. In other studies, expression of heterologous mRNA in oocytes has led apparently to detection of inhibitors as well as activators of amino acid transport. Some amino acid transport systems also could conceivably contain nucleic acid as well as glycoprotein components.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Transport Systems↗
Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic element.
System A amino acid transporter (SNAT2) gene expression is up-regulated at the transcriptional level in response to amino acid deprivation. Functional analysis of genomic fragments 5' upstream of the transcription start site, for both human and mouse SNAT2 genes showed that these regions exhibit promoter activity, but were amino acid unresponsive. However, when the human and mouse constructs were extended to include intron 1, it was observed that the rate of transcription was increased following amino acid deprivation. Deletion analysis of the human gene identified an intron 1 sequence spanning 54 nucleotides that was sufficient for conferring amino acid-dependent regulation to a minimal SNAT2 promoter. Alignment of the corresponding region from the human, mouse, and rat genomes revealed three highly conserved sequences. From site-directed mutagenesis, it was concluded that one of these sites functions as an amino acid response element (AARE) to regulate transcription. The core sequence of this site is identical to the AARE in the human CHOP gene. The SNAT2 AARE, along with a nearby conserved CAAT box, has enhancer activity in that it functions in an orientation and position independent manner, and it confers regulated transcription to a heterologous promoter.
Amino Acid Transport System A↗
Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.
The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.
Biological Transport, Active↗
Regulation of amino acid transport system L by amino acid availability in CHO-K1 cells. A special role for leucine.
Starvation of CHO-K1 cells for leucine leads to a 3-4-fold increase in transport system L activity, without modification of transport through systems A and ASC. The concentration of leucine must be below 10 microM before the enhancement of transport can be clearly seen. To achieve low concentrations of leucine such as 10 microM, extensive dialysis of fetal calf serum was required. The enhancement of transport was completed after 12-24 h of starvation and was fully reversed within 1 h of re-feeding with leucine. Starvation for isoleucine, valine or phenylalanine also produced an increase in system L transport activity, but the effect was only one half of that seen following leucine starvation.
Amino Acids↗
Characterization of an N-system amino acid transporter expressed in retina and its involvement in glutamine transport.
We report here on the characterization of a mouse N-system amino acid transporter protein, which is involved in the transport of glutamine. This protein of 485 amino acids shares 52% sequence homology with an N-system amino acid transporter, mouse N-system amino acid transporter (mNAT) and its orthologs. Because this protein shares a high degree of sequence homology and functional similarity to mNAT, we named it mNAT2. mNAT2 is predominately expressed in the retina and to a slightly lesser extent in the brain. In the retina, it is located in the axons of ganglion cells in the nerve fiber layer and in the bundles of the optic nerve. Functional analysis of mNAT2 expressed in Xenopus oocytes revealed that the strongest transport activities were specific for l-glutamine. In addition, mNAT2 is a Na(+)- and pH-dependent, high affinity transporter and partially tolerates substitution of Na(+) by Li(+). Additionally, mNAT2 functions as a carrier-mediated transporter that facilitates efflux. The unique expression pattern and selective glutamine transport properties of mNAT2 suggest that it plays a specific role in the uptake of glutamine involved in the generation of the neurotransmitter glutamate in retina.
Acetyltransferases↗
Evidence for a close link between the thyroid hormone transport system and the aromatic amino acid transport system T in erythrocytes.
The transport of [125I]triiodothyronine ([125I]T3) and [3H]tryptophan ([3H]Trp) by washed rat erythrocytes was studied at 25 degrees C in the presence of leucine in order to block the neutral amino acid transport system L. Eadie-Hofstee plots of initial velocity data gave the following values of Km (micromolar) and Vmax (nanomole/min/10(8) cells): 0.122 +/- 0.007 and 0.140 +/- 0.021 for T3, and 558 +/- 28 and 17.4 +/- 2.3 for Trp (n = 5). The Trp transport system in rat erythrocytes is similar to the human erythrocyte aromatic amino acid-specific system T described by Rosenberg et al. (Rosenberg, R., Young, J. D., and Ellory, J. C. (1980) Biochim. Biophys. Acta 598, 375-384). Unlabeled aromatic amino acids (e.g. Trp, phenylalanine, tyrosine) competitively inhibited [125I]T3 uptake and unlabeled iodothyronine analogues (e.g. T3, D-T3, thyroxine, thyronine) competitively inhibited [3H]Trp uptake. The inhibition constants of these competitors measured with each labeled substrate were highly correlated. N-Ethylmaleimide irreversibly inhibited T3 and Trp transport and each substrate protected the transport system of the other from inactivation by N-ethylmaleimide. The Vmax of T3 and Trp transport by human erythrocytes were 500 and 120 times lower, respectively, than those of rat erythrocytes (0.30 and 126 pmol/min/10(8) cells, respectively). The T3 and Trp transport activities of sheep erythrocytes were undetectable. These results indicate that T3 and Trp either share a common multi-specific transport system or are transported by closely linked systems which interact in the erythrocyte membrane.
Animals↗
The amino acid transport system b(o,+) and cystinuria.
Amino acid transport in mammalian plasma membranes is mediated by a multiplicity of amino acid transport systems. Some of them (systems L, y+ L, x(c)- and b(o,+)) are the result of the activity of heteromeric amino acid transporters (HAT) (i.e. transport activity is elicited by the coexpression of a heavy and a light subunit). The two heavy subunits known today (HSHAT: rBAT and 4F2hc) were identified in 1992, and light subunits (LSHAT: LAT-1, LAT-2, asc-1, y+ LAT-1, y+ LAT-2, xCT and b(o,+)AT) have been cloned in the last 2 years. Defects in two genes of this family (SLC3A1, encoding rBAT and SLC7A9, encoding b(o,+)AT) are responsible for cystinuria, an inherited aminoaciduria of cystine and dibasic amino acids. This finding and functional studies of rBAT and b(o,+)AT suggested that these two proteins encompassed the high-affinity renal reabsorption system of cystine. In contrast to this view, immunofluorescence studies showed that rBAT is most abundant in the proximal straight tubule, and b(o,+)AT is most abundant in the proximal convoluted tubule of the nephron. The need for a new light subunit for rBAT and a heavy subunit for b(o,+)AT is discussed.
Amino Acid Transport Systems↗
Heterotropic effects of dipolar amino acids on the activity of the anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles.
D-Aspartic acid was used as a specific substrate to evaluate the effects of dipolar amino acids on the high affinity anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles. At pH 6, increasing L-phenylalanine concentrations caused a saturable activation of 0.05 mM D-aspartic acid uptake (Ka = 2.4 mM), and a saturating concentration of effector increased the Vmax of transport (2.6-fold) without any significant effect on the Km. At pH 8, however, a complex activation/inhibition curve was obtained with increasing L-phenylalanine concentrations, and a saturating concentration of effector increased both the Vmax (1.5-fold) and Km (2.1-fold) for transport. Increasing concentrations of L-valine, L-isoleucine, L-methionine, and L-threonine also showed complex activation/inhibition curves of D-aspartic acid uptake at both pH 6 and 8. The maximum level of activation, the plateau reached at saturating concentrations, and the concentration of effector producing either maximum activation or inhibition were, however, different at these two pH values. By using an optimum concentration of 10 mM L-valine at pH 6, the absence of trans-activation and of further activation by a cis-gradient of effector could be demonstrated. These results suggest that two allosteric sites directly accessible from the external medium are responsible for the heterotropic activation of intestinal system X-AG by dipolar amino acids and that, under physiological conditions, such effects might compensate for the lack of specificity of the neutral brush-border system.
Amino Acid Transport Systems↗
Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction.
Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using alpha(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 x essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.
Amino Acid Transport System A↗
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+.
Amino Acid Transport Systems↗
Epithelial cells isolated from chicken jejunum: an experimental model for the study of the functional properties of amino acid transport system b(0,+).
The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b(0,+) activity, the broad-spectrum and Na(+)-independent amino acid transporter. The half-saturation constant for lysine entry (K(m)+/-S.E.) was 0.029+/-0.002 mM and the flux was not affected significantly by Na(+) replacement with choline. Lysine influx was inhibited by L-leucine both in Na(+) and choline medium with inhibition constants (K(i)+/-S.E.) 0.068+/-0.006 mM (in Na(+)) and 0.065+/-0.009 mM (in choline). Other inhibitory amino acids (K(i)+/-S.E.) were (mM): L-tyrosine (0.073+/-0.018), L-methionine (0.15+/-0.015), L-cystine (0.42+/-0.04), L-cysteine (1.1+/-0.07), L-isoleucine (1.1+/-0.09), L-glutamine (1.8+/-0.16) and L-valine (2.5+/-0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM L-lysine and L-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y(+)L, the broad-spectrum and cation-modulated transporter.
Amino Acid Transport Systems↗
Expression of the mammalian Na+-independent L system amino acid transporter in Xenopus laevis oocytes.
System L is primarily responsible for the Na+-independent transport of neutral amino acids, those with bulky chains such as leucine, isoleucine, phenylalanine, etc., into mammalian cells. mRNA from rat kidney and human lymphoid cells, when microinjected into Xenopus laevis oocytes, induced expression of this transport system. The expressed transport exhibits characteristics similar to those reported for the System L amino acid transporter from a variety of mammalian cells. Injection of size-fractionated mRNA indicates that the System L transporter in both the rat kidney and human lymphoid cells is encoded by mRNA of about 3 to 4 kb.
Amino Acid Transport Systems↗
Glial induction of blood-brain barrier-like L-system amino acid transport in the ECV304 cell line.
The blood-brain barrier (BBB) is formed by the presence of tight junction complexes between brain endothelial cells that restrict paracellular permeability. As a consequence, a number of transport proteins are expressed on cerebral endothelial cells to facilitate the transport of nutrients into the brain. Although the modulation of barrier tight junction properties by glial-conditioned medium and by second messengers is well established, little is known about the effects of these factors on carrier-mediated BBB transport processes. The ECV304 cell line shows an endothelial phenotype and can be induced to upregulate certain BBB features in the presence of glial factors. In the present study, we have examined the effect of conditioned medium derived from rat C6-glioma cells (C6CM) on the function of the L-system amino acid transporter in ECV304 cells, using L-leucine as the model substrate, and have determined whether the changes observed can be mimicked by modulating intracellular cAMP levels. ECV304 cells exposed to C6CM exhibited a significant increase in both the affinity of leucine transport and the diffusional constant (Michaelis-Menten), while the maximal transport capacity remained unchanged. Conversely, acute exposure to modulators of the PKA and PKC second messenger pathways was found to reduce significantly the maximal transport capacity and diffusion constants, while transport affinity remained unchanged. In both cases, the maximal flux of leucine was increased, indicating transport of greater efficiency. This study indicates that exposure of ECV304 cells to C6CM provides an influence inducing L-system transport properties characteristic of brain endothelial cells. Furthermore, it appears that L-system-mediated transport of amino acids can be modulated by several distinct pathways.
Amino Acid Transport System L↗
Amino acid transport system A resembles system N in sequence but differs in mechanism.
Classical amino acid transport System A accounts for most of the Na(+)-dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short- and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling.
Amino Acid Sequence↗
Kinetic properties, nutrient-dependent regulation and energy coupling of amino-acid transport systems in Penicillium cyclopium.
In submerged grown hyphae of Penicillium cyclopium the activities of seven transport systems could be distinguished which share in the uptake of L-arginine, L-glutamic acid, L-phenylalanine and L-leucine. They include the specific systems a (accepting L-arginine and L-lysine), b (L-phenylalanine, L-tyrosine), c (L-glutamic acid) and d (L-leucine), system I (a 'general amino-acid permease') and the low-affinity systems II and III, which accept acidic or basic amino acids, respectively, but also L-phenylalanine. In nutrient-sufficient cells, systems I, II and III remain repressed; uptake is dominated by the specific systems b, c, d and a, the latter reaching its maximum activity. Nitrogen starvation is the most powerful signal for the development of systems I, II and III, whereas, in carbon-starved cells, systems b, c and d reach maximum activities. The development of the general amino-acid permease in nitrogen-starved cells requires both translational and--with a few hours delay--transcriptional events as indicated by the influence of cycloheximide and 5-fluorouracil. The uptake of all amino acids is accompanied by a transient acidification of the cellular interior. Short-time preaccumulation of several anions, such as citrate, alpha-oxo-glutarate, glutamate (but not glutamine), increases the initial rate of amino-acid uptake at a pH above the optimum. Uncouplers inhibit the uptake not only under aerobic but also under anaerobic conditions, where the ATP content is not influenced by these compounds. These findings point to an H+/amino acid symport, which is tightly connected with the recycling of the incoming protons by the plasmalemma H+-ATPase.
Adenosine Triphosphate↗