Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanism(s) mediating [14C]L-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that L-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]L-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with L-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

Physiology and pathology of neutral amino acid transport in renal and intestinal epithelial cells

About 95% of nutrient protein is absorbed in the mammalian intestine as amino acids and trl-and dipeptides. Once absorbed by the intestine amino acids are distributed by the circulation throughout the body. In the kidney an ultrafiltrate of the blood plasma is generated from which all amino acids are reabsorbed in the proximal tubule. System B{u2070} is the main amino acid transport system in these two tissues for broad neutral amino acids. Four transporters B{u2070}AT1 (SLC6A19), B{u2070}AT2 (SLC6A15), B{u2070}AT3 (SLC6A18) and ASTC2 (SCL1A5) have properties of this system when studied in isolation. B{u2070}ATl and B{u2070}AT2 co-transport one Na{u207A} -ion and one amino acid substrate. The low-affinity B{u2070}ATl transporter accepts most neutral amino acids, while the high-affinity transporter B{u2070}AT2 prefers branch-chained amino acids and proline. B{u2070}AT3 prefers alanine and glycine, but has also been reported to transport a variety of neutral amino acids. ASCT2 is a Na{u207A}-dependent antiporter, preferring neutral amino acids except those with an aromatic side-chain. However, in two studies it has been reported that amino acid transport of ASCT2 was inhibited also by phenylalanine. Analysis of ASCT2 function in vivo and in vitro, has led to the proposal that it represents the molecular correlate of system B{u2070} in kidney and intestine. Mutations in B{u2070}ATl, however, cause Hartnup disorder, a defect of neutral amino acid transport in kidney and intestine. Thus it is important to reconcile differences and to clarify the contribution of these amino acid transporters to system B{u2070}-like transport in kidney and intestine. Therefore, the overall aim for this thesis was to investigate the distribution and contribution of B{u2070}AT1 (SlC6A19), B{u2070}AT2 (SlC6A15), and ASTC2 (SCL1AS) to neutral amino acid transport in the kidney. Immunofluorescence studies revealed localisation of B{u2070}AT1 in the apical membrane of early (S1-S2) segments of the proximal tubule, while SOAT2 was localised to the apical membrane of the later (S2-S3) segments. This is consistent with physiological data reporting low-affinity transport in early segments and high-affinity transport in later segments of the proximal tubule. ASCT2 was localised in the apical membrane of S2 segments and in the basolateral membrane of the distal tubule. Transport studies with renal brush border membrane vesicles revealed a dominant transport activity that is consistent with the properties of B{u2070}AT1. This transport activity was lacking in B{u2070}AT1-deficient mice. B{u2070}AT2 activity was excluded by competition studies with selective substrates. ASCT2 activity was excluded due to the lack of additional amino acid transport in brush border membrane vesicles preloaded with A5CT2 amino acid substrates. Therefore, it is concluded that B{u2070}AT2 and ASCT2 do not contribute significantly to neutral amino acid transport in the kidney. B{u2070}AT1 is the main transport activity for neutral amino acid transport in the kidney

N-System Amino Acid Transport at the Blood-CSF Barrier

Despite l-glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l-[ 14 C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na + -dependent mechanism. The Na + -independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na + -dependent uptake was inhibited neither by the A-system substrate Α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l-glutamate Γ-hydroxamate, three N-system substrates. Replacement of Na + with Li + had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V max and K max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66421/1/j.1471-4159.1995.65062571.x.pd

Identification of a membrane protein, LAT-2, that co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta >> brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells

Pathways of L-glutamic acid transport in cultured human fibroblasts.

The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax

Cloning and expression of a novel Na(+)-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters

A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT\u27s apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines

Thyroxine-Induced Changes in the Development of Neutral Α-Amino Acid Transport Systems of Rat Brain

Transport of representative neutral Α-amino acids was measured in brain slices after injecting thy-roxine into donor rats of various ages from 1 to 23 days old. The hormone did not alter uptake in slices from 1-day-old rats even when treatment was begun on pregnant rats as much as 10 days before delivery. Injecting thy-roxine until age 6 days, however, decreased the activity of transport system A (the major sodium-dependent system in most mammalian cells) and caused appearance of a new transport system used by the model amino acids, 2-aminoisobutyric acid and 2-(methylamino)isobutyric acid. Uptake at 6 days was similar to that found in slices from older, untreated rats (e.g., those 14 days old). These results strongly suggest that one action of thyroxine is to accelerate the development of neutral Α-amino acid transport systems of brain over the first six days after birth. Thyroxine treatment of rats from birth to age 14 days also appears to increase the activities of both system A and the second transport system used by the two model amino acids in brains from 14-day-old rats.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66252/1/j.1471-4159.1984.tb02781.x.pd

Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle

AbstractTo date, two different transporters that are capable of transporting α-(methylamino)isobutyric acid, the specific substrate for amino acid transport system A, have been cloned. These two transporters are known as ATA1 and ATA2. We have cloned a third transporter that is able to transport the system A-specific substrate. This new transporter, cloned from rat skeletal muscle and designated rATA3, consists of 547 amino acids and has a high degree of homology to rat ATA1 (47% identity) and rat ATA2 (57% identity). rATA3 mRNA is present only in the liver and skeletal muscle. When expressed in Xenopus laevis oocytes, rATA3 mediates the transport of α-[14C](methylamino)isobutyric acid and [3H]alanine. With the two-microelectrode voltage clamp technique, we have shown that exposure of rATA3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. The amino acid-induced current is Na+-dependent and pH-dependent. Analysis of the currents with alanine as the substrate has shown that the K0.5 for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2±0.1 mM and that the Na+:alanine stoichiometry is 1:1

ASCT-1 Is a Neutral Amino Acid Exchanger with Chloride Channel Activity

The ubiquitous transport activity known as system ASC is characterized by a preference for small neutral amino acids including alanine, serine, and cysteine. ASCT-1 and ASCT-2, recently cloned transporters exhibiting system ASC-like selectivity, are members of a major amino acid transporter family that includes a number of glutamate transporters. Here we show that ASCT1 functions as an electroneutral exchanger that mediates negligible net amino acid flux. The electrical currents previously shown to be associated with ASCT1-mediated transport result from activation of a thermodynamically uncoupled chloride conductance with permeation properties similar to those described for the glutamate transporter subfamily. Like glutamate transporters, ASCT1 activity requires extracellular Na+. However, unlike glutamate transporters, which mediate net flux and complete a transport cycle by countertransport of K+, ASCT-1 mediates only homo- and heteroexchange of amino acids and is insensitive to K+. The properties of ASCT-1 suggest that it may function to equilibrate different pools of neutral amino acids and provide a mechanism to link amino acid concentration gradient

Cloning and functional characterization of a Na+-independent, broad-specific neutral amino acid transporter from mammalian intestine

AbstractWe have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with Kt values in the range of 100–1000 μM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (Kt values in the range of 10–20 μM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution