scholarly journals Structural Insights Into Thyroid Hormone Transport Mechanisms of the L-Type Amino Acid Transporter 2

2015 ◽  
Vol 29 (6) ◽  
pp. 933-942 ◽  
Author(s):  
Katrin M. Hinz ◽  
Katja Meyer ◽  
Anita Kinne ◽  
Ralf Schülein ◽  
Josef Köhrle ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Competitive Inhibition ◽  
Expression System ◽  
Transmembrane Helix ◽  
Amino Acid Transporter ◽  
Recognition Site ◽  
Side Chain ◽  
Xenopus Laevis Oocytes ◽  
Acid Transporter

Abstract Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3′-diiodothyronine (3,3′-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3′-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3′-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport.

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 Multiple components of arginine and phenylalanine transport induced in neutral and basic amino acid transporter-cRNA-injected Xenopus oocytes

1996 ◽  
Vol 318 (3) ◽  
pp. 915-922 ◽  
Author(s):  
George J PETER ◽  
Iain G. DAVIDSON ◽  
Aamir AHMED ◽  
Lynn McILROY ◽  
Alexander R. FORRESTER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Competitive Inhibition ◽  
Protein Complexes ◽  
Basic Amino Acid ◽  
Amino Acid Transporter ◽  
Transport Processes ◽  
Arginine Transport ◽  
Acid Transporter ◽  
Phenylalanine Transport

The induced uptakes of l-[3H]phenylalanine and l-[3H]arginine in oocytes injected with clonal NBAT (neutral and basic amino acid transporter) cRNA show differential inactivation by pre-treatment with N-ethylmaleimide (NEM), revealing at least two distinct transport processes. NEM-resistant arginine transport is inhibited by leucine and phenylalanine but not by alanine or valine; mutual competitive inhibition of NEM-resistant uptake of arginine and phenylalanine indicates that the two amino acids share a single transporter. NEM-senstive arginine transport is inhibited by leucine, phenylalanine, alanine and valine. At least two NEM-sensitive transporters may be expressed because we have been unable to confirm mutual competitive inhibition between arginine and phenylalanine transport. The NEM-resistant transport mechanism appears to involve distinct but overlapping binding sites for cationic and zwitterionic substrates. NBAT is known to form oligomeric protein complexes in cell membranes, and its functional roles when expressed in Xenopus oocytes may include interaction with oocyte proteins, leading to increased native amino acid transport activities; these resemble NBAT-expressed activities in terms of NEM-sensitivity and apparent substrate range (including an unusual inhibition by β-phenylalanine).

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 Transport of butyryl-l-carnitine, a potential prodrug, via the carnitine transporter OCTN2 and the amino acid transporter ATB0,+

2007 ◽  
Vol 293 (5) ◽  
pp. G1046-G1053 ◽  
Author(s):  
Sonne R. Srinivas ◽  
Puttur D. Prasad ◽  
Nagavedi S. Umapathy ◽  
Vadivel Ganapathy ◽  
Prem S. Shekhawat
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
High Capacity ◽  
Amino Acid Transporter ◽  
Xenopus Laevis Oocytes ◽  
Loss Of Function ◽  
Bacterial Fermentation ◽  
Carnitine Transporter ◽  
Inward Currents ◽  
Acid Transporter

l-Carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB0,+. Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB0,+ is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-l-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB0,+ to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB0,+ and OCTN2 in heterologous expression systems. BC inhibited ATB0,+-mediated glycine transport in mammalian cells (IC50, 4.6 ± 0.7 mM). In Xenopus laevis oocytes expressing human ATB0,+, BC induced Na+-dependent inward currents under voltage-clamp conditions. The currents were saturable with a K0.5 of 1.4 ± 0.1 mM. Na+ activation kinetics of BC-induced currents suggested involvement of two Na+ per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC50, 1.5 ± 0.3 μM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na+ dependent and saturable ( K0.5, 0.40 ± 0.02 μM). We conclude that ATB0,+ is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB0,+ may mediate intestinal absorption of BC when OCTN2 is defective.

Expression of the mammalian Na+-independent L System amino acid transporter in Xenopus laevis oocytes

1989 ◽  
Vol 275 (2) ◽  
pp. 591-596 ◽  
Author(s):  
Suresh S. Tate ◽  
Reiko Urade ◽  
Thomas V. Getchell ◽  
Sidney Udenfriend
Keyword(s):  
Amino Acid ◽  
Xenopus Laevis ◽  
Amino Acid Transporter ◽  
Xenopus Laevis Oocytes ◽  
L System ◽  
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 Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12

2004 ◽  
Vol 186 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Takashi Koyanagi ◽  
Takane Katayama ◽  
Hideyuki Suzuki ◽  
Hidehiko Kumagai
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Competitive Inhibition ◽  
Binding Protein ◽  
Amino Acid Transporter ◽  
Binding Studies ◽  
E Coli ◽  
Acid Transporter ◽  
Branched Chain ◽  
K 12

ABSTRACT In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (ΔaroP ΔpheP Δmtr Δtna ΔtyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The Km values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 μM, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.

scholarly journals Constitutive Endocytosis of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter-3 Requires ARFGAP1

2021 ◽  
Vol 12 ◽  
Author(s):  
Kusumika Saha ◽  
Jae-Won Yang ◽  
Tina Hofmaier ◽  
SanthoshKannan Venkatesan ◽  
Thomas Steinkellner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Excitatory Amino Acid ◽  
Expression System ◽  
Amino Acid Transporter ◽  
Endocytic Pathway ◽  
Excitatory Amino Acid Transporter ◽  
C Terminus ◽  
Acid Transporter

The eukaryotic endocytic pathway regulates protein levels available at the plasma membrane by recycling them into specific endosomal compartments. ARFGAP1 is a component of the coat protein I (COPI) complex but it also plays a role in promoting adapter protein-2 (AP-2) mediated endocytosis. The excitatory amino acid transporter-3 (EAAT3) mediates the reuptake of glutamate from the synaptic cleft to achieve rapid termination of synaptic transmission at glutamatergic synapses. In this study, we identified two interacting proteins of EAAT3 by mass spectrometry (MS) ARFGAP1 and ARF6. We explored the role of ARFGAP1 and ARF6 in the endocytosis of EAAT3. Our data revealed that ARFGAP1 plays a role in the recycling of EAAT3, by utilizing its GTPase activating protein (GAP) activity and ARF6 acting as the substrate. ARFGAP1 promotes cargo sorting of EAAT3 via a single phenylalanine residue (F508) located at the C-terminus of the transporter. ARFGAP1-promoted AP-2 dependent endocytosis is abolished upon neutralizing F508. We utilized a heterologous expression system to identify an additional motif in the C-terminus of EAAT3 that regulates its endocytosis. Impairment in endocytosis did not affect somatodendritic targeting in cultured hippocampal neurons. Our findings support a model where endocytosis of EAAT3 is a multifactorial event regulated by ARFGAP1, occurring via the C-terminus of the transporter, and is the first study to examine the role of ARFGAP1 in the endocytosis of a transport protein.

Expression of the amino acid transporter ATB0+in lung: possible role in luminal protein removal

2003 ◽  
Vol 284 (1) ◽  
pp. L39-L49 ◽  
Author(s):  
Jennifer L. Sloan ◽  
Barbara R. Grubb ◽  
Sela Mager
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Short Circuit ◽  
Amino Acid Transporter ◽  
Normal Lung ◽  
Alveolar Type ◽  
Type I ◽  
Western Blots ◽  
Acid Transporter

Normal lung function requires transepithelial clearance of luminal proteins; however, little is known about the molecular mechanisms of protein transport. Protein degradation followed by transport of peptides and amino acids may play an important role in this process. We previously cloned and functionally characterized the neutral and cationic amino acid transporter ATB0+and showed expression in the lung by mRNA analysis. In this study, the tissue distribution, subcellular localization, and function of the transporter in native tissue were investigated. Western blots showed expression of the ATB0+protein in mouse lung, stomach, colon, testis, blastocysts, and human lung. Immunohistochemistry revealed that ATB0+is predominantly expressed on the apical membrane of ciliated epithelial cells throughout mouse airways from trachea to bronchioles and in alveolar type I cells. Electrical measurements from mouse trachea preparations showed Na+- and Cl−-dependent, amino acid-induced short-circuit current consistent with the properties of ATB0+. We hypothesize that, by removing amino acids from the airway lumen, the transporter contributes to protein clearance and, by maintaining a low nutrient environment, plays a role in lung defense.

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