Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids

2017 ◽  
Vol 443 ◽  
pp. 163-174 ◽  
Author(s):  
Katrin M. Hinz ◽  
Dominik Neef ◽  
Claudia Rutz ◽  
Jens Furkert ◽  
Josef Köhrle ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Thyroid Hormones ◽  
Amino Acid Transporter ◽  
Molecular Features ◽  
Import And Export ◽  
Acid Transporter

scholarly journals Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

2000 ◽  
Vol 346 (3) ◽  
pp. 705-710 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Anion Channel ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Anion Conductance ◽  
Neutral Amino Acid Transporter ◽  
Inward Currents ◽  
Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

scholarly journals Intestinal peptidases form functional complexes with the neutral amino acid transporter B0AT1

2012 ◽  
Vol 446 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Stephen J. Fairweather ◽  
Angelika Bröer ◽  
Megan L. O'Mara ◽  
Stefan Bröer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Amino Acid Transporter ◽  
The Body ◽  
Site Directed Mutagenesis ◽  
Substrate Affinity ◽  
Xenopus Laevis Oocytes ◽  
Acid Transporter

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

scholarly journals Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Doreen Schlisselberg ◽  
Eldar Mazarib ◽  
Ehud Inbar ◽  
Doris Rentsch ◽  
Peter J. Myler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Transporter ◽  
Acid Transporter

scholarly journals Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3′-Diiodothyronine across the Plasma Membrane

2015 ◽  
Vol 4 (Suppl. 1) ◽  
pp. 42-50 ◽  
Author(s):  
Anita Kinne ◽  
Melanie Wittner ◽  
Eva K. Wirth ◽  
Katrin M. Hinz ◽  
Ralf Schülein ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thyroid Hormone ◽  
Thyroid Hormones ◽  
Surface Expression ◽  
Amino Acid Transporter ◽  
Cell System ◽  
Monocarboxylate Transporter ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
Acid Transporter

Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of Lat2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse Lat2. The coinjection of cRNA coding for Lat2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3′-diiodo-L-thyronine (3,3′-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by Lat2, we inhibited 3,3′-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid strongly competed with 3,3′-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both Lat2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by Lat2. Our data indicates that Lat2 compared to other thyroid hormone transporters prefers 3,3′-T2 as the substrate. Thus, Lat2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3′-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.

scholarly journals The zebrafish cationic amino acid transporter/glycoprotein-associated family: sequence and spatiotemporal distribution during development of the transport system b0,+ (slc3a1/slc7a9)

2021 ◽  
Author(s):  
Ståle Ellingsen ◽  
Shailesh Narawane ◽  
Anders Fjose ◽  
Tiziano Verri ◽  
Ivar Rønnestad
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transport System ◽  
Expression Patterns ◽  
Amino Acid Transporter ◽  
Rapid Screening ◽  
Cationic Amino Acid ◽  
Large Neutral Amino Acids ◽  
Neutral Amino Acids ◽  
Acid Transporter

AbstractSystem b0,+ absorbs lysine, arginine, ornithine, and cystine, as well as some (large) neutral amino acids in the mammalian kidney and intestine. It is a heteromeric amino acid transporter made of the heavy subunit SLC3A1/rBAT and the light subunit SLC7A9/b0,+AT. Mutations in these two genes can cause cystinuria in mammals. To extend information on this transport system to teleost fish, we focused on the slc3a1 and slc7a9 genes by performing comparative and phylogenetic sequence analysis, investigating gene conservation during evolution (synteny), and defining early expression patterns during zebrafish (Danio rerio) development. Notably, we found that slc3a1 and slc7a9 are non-duplicated in the zebrafish genome. Whole-mount in situ hybridization detected co-localized expression of slc3a1 and slc7a9 in pronephric ducts at 24 h post-fertilization and in the proximal convoluted tubule at 3 days post-fertilization (dpf). Notably, both the genes showed co-localized expression in epithelial cells in the gut primordium at 3 dpf and in the intestine at 5 dpf (onset of exogenous feeding). Taken together, these results highlight the value of slc3a1 and slc7a9 as markers of zebrafish kidney and intestine development and show promise for establishing new zebrafish tools that can aid in the rapid screening(s) of substrates. Importantly, such studies will help clarify the complex interplay between the absorption of dibasic amino acids, cystine, and (large) neutral amino acids and the effect(s) of such nutrients on organismal growth.

ACE2 and gut amino acid transport

2020 ◽  
Vol 134 (21) ◽  
pp. 2823-2833 ◽  
Author(s):  
Simone M.R. Camargo ◽  
Raphael N. Vuille-dit-Bille ◽  
Chantal F. Meier ◽  
François Verrey
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Amino Acid ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Amino Acid Transporters ◽  
Type I ◽  
Small Intestinal Mucosa ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

Abstract ACE2 is a type I membrane protein with extracellular carboxypeptidase activity displaying a broad tissue distribution with highest expression levels at the brush border membrane (BBM) of small intestine enterocytes and a lower expression in stomach and colon. In small intestinal mucosa, ACE2 mRNA expression appears to increase with age and to display higher levels in patients taking ACE-inhibitors (ACE-I). There, ACE2 protein heterodimerizes with the neutral amino acid transporter Broad neutral Amino acid Transporter 1 (B0AT1) (SLC6A19) or the imino acid transporter Sodium-dependent Imino Transporter 1 (SIT1) (SLC6A20), associations that are required for the surface expression of these transport proteins. These heterodimers can form quaternary structures able to function as binding sites for SARS-CoV-2 spike glycoproteins. The heterodimerization of the carboxypeptidase ACE2 with B0AT1 is suggested to favor the direct supply of substrate amino acids to the transporter, but whether this association impacts the ability of ACE2 to mediate viral infection is not known. B0AT1 mutations cause Hartnup disorder, a condition characterized by neutral aminoaciduria and, in some cases, pellagra-like symptoms, such as photosensitive rash, diarrhea, and cerebellar ataxia. Correspondingly, the lack of ACE2 and the concurrent absence of B0AT1 expression in small intestine causes a decrease in l-tryptophan absorption, niacin deficiency, decreased intestinal antimicrobial peptide production, and increased susceptibility to inflammatory bowel disease (IBD) in mice. Thus, the abundant expression of ACE2 in small intestine and its association with amino acid transporters appears to play a crucial role for the digestion of peptides and the absorption of amino acids and, thereby, for the maintenance of structural and functional gut integrity.

scholarly journals Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2

2010 ◽  
Vol 298 (6) ◽  
pp. C1401-C1413 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
Catia Sternini ◽  
Enrique Rozengurt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Isobutyric Acid ◽  
Amino Acid Transporter ◽  
Membrane Depolarization ◽  
Neutral Amino Acid ◽  
Acid Transport ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na+-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ signaling in STC-1 cells was suppressed by removing Na+ from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

scholarly journals Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP-Encoded Transporter of Escherichia coli

2009 ◽  
Vol 76 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Yurgis A. V. Yomantas ◽  
Irina L. Tokmakova ◽  
Natalya V. Gorshkova ◽  
Elena G. Abalakina ◽  
Svetlana M. Kazakova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Target Genes ◽  
Aromatic Amino Acids ◽  
Amino Acid Transporter ◽  
Methylotrophic Bacteria ◽  
Methylophilus Methylotrophus ◽  
Acid Transporter

ABSTRACT The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Kmr marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.

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