Conserved tyrosine-147 plays a critical role in the ligand-gated current of the epithelial cation/amino acid transporter/channel CAATCH1

SUMMARYCAATCH1 functions both as an amino-acid-gated cation channel and as a cation-dependent, proline-preferring, nutrient amino acid transporter in which the two functions are thermodynamically uncoupled. This study focuses on the ionic channel aspect, in which a Tyr147 (wild type) to Phe147 (Y147F) site-directed mutation was investigated by steady-state electrophysiological measurements in the Xenopus laevisoocyte expression system. This tyrosine residue is conserved within the third transmembrane domain in members of the Na+:neurotransmitter transporter family (SNF), where it plays a role in binding pharmacological ligands such as cocaine to the serotonin (SERT), dopamine (DAT) and norepinephrine (NET) transporters. Epithelial CAATCH1 is a member of the SNF family. The results show that amino acid ligand-gating selectivity and current magnitudes in Na+- and K+-containing media are differentially altered in CAATCH1 Y147F compared with the wild type. In the absence of amino acid ligands, the channel conductance of Na+,K+ and Li+ that is observed in the wild type was reduced to virtually zero in Y147F. In the wild type, proline binding increased conductance strongly in Na+-containing medium and moderately in K+-containing medium, whereas in Y147F proline failed to elicit any cation currents beyond those of N-methyl-D-glucamine- or water-injected oocytes. In the wild type, methionine binding strongly inhibited inward Na+ currents, whereas in Y147F it strongly stimulated inward currents in both Na+ and K+-containing media. Indeed, in Na+-containing medium, the relative potency ranking for inward current inhibition in the wild type(Met>Leu>Gly>Phe>Thr) was similar to the ranking of ligand-permissive gating of large inward currents in Y147F. In Na+-containing medium, current/voltage relationships elicited by ligands in the wild type were complex and reversing, whereas in Y147F they were linear and inwardly rectifying. In K+-containing medium,current/voltage relationships remained non-linear in Y147F. Both wild-type and Y147F currents were Cl--independent. Together, these data demonstrate a critical role for Tyr147 in ligand-binding selectivity and modulation of the ionic channel conductance in CAATCH1. The results support the argument that inhibition of the CAATCH1 conductance by free methionine shares some properties in common with ligand inhibition of DAT, SERT, NET and the γ-aminobutyric acid transporter (GAT1).

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

Biochemical Journal ◽

10.1042/bj3460705 ◽

2000 ◽

Vol 346 (3) ◽

pp. 705-710 ◽

Cited By ~ 58

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+.

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00233.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. G1046-G1053 ◽

Cited By ~ 37

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.

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Tadr Is an Axonal Histidine Transporter Required for Visual Neurotransmission in Drosophila

10.1101/2021.12.01.470757 ◽

2021 ◽

Author(s):

Yongchao Han ◽

Lei Peng ◽

Tao Wang

Keyword(s):

Amino Acid ◽

Synaptic Transmission ◽

Histidine Decarboxylase ◽

De Novo ◽

Critical Role ◽

Amino Acid Transporter ◽

Monoamine Neurotransmitter ◽

Monoamine Neurotransmitters ◽

Specific Amino Acid ◽

Acid Transporter

AbstractNeurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by Histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. Here, we identified a specific histidine transporter, TADR (Torn And Diminished Rhabdomeres), that is required for visual transmission in Drosophila. TADR and Hdc co-localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission.

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The cationic amino acid exporter Slc7a7 is vital for and induced in tissue macrophages with sustained efferocytic activity

10.1101/2020.04.20.051664 ◽

2020 ◽

Author(s):

Doris Lou Demy ◽

Mireille Carrère ◽

Ramil Noche ◽

Muriel Tauzin ◽

Marion Le Bris ◽

...

Keyword(s):

Amino Acid ◽

Neuronal Apoptosis ◽

Amino Acid Transporter ◽

Cationic Amino Acid Transporter ◽

Adaptive Responses ◽

Wild Type ◽

Functional Link ◽

Cationic Amino Acid ◽

Acid Transporter ◽

High Level

ABSTRACTMost tissues harbor a substantial population of resident macrophages. It is not quite known yet how their quite diverse phenotypes are shaped by the functions that they assume in each tissue. In this study, we elucidate a functional link between the Slc7a7 cationic amino acid transporter and tissue macrophages. We had identified a mutant zebrafish devoid of microglia due to a mutation in the slc7a7 gene. We found that in Slc7a7 deficient larvae, macrophages do enter the retina and brain to become microglia, but then die during the developmental wave of neuronal apoptosis, which triggers intense efferocytic work from them. A similar macrophage demise occurs at other tissues and stages whereby macrophages have to engulf many cell corpses, be it due to developmental or experimentally triggered cell death. We found that slc7a7 is by far the main cationic amino acid transporter gene expressed in macrophages of wild type zebrafish larvae, and that its expression is induced in tissue macrophages within 1-2 hrs upon efferocytosis. Our data altogether indicate that a high level of Slc7a7 is vital not only for microglia but also for any steadily efferocytic tissue macrophages, and that slc7a7 gene induction is one of the adaptive responses that allow them to cope with the catabolism of numerous dead cells without compromising their own viability.

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Mutations of the basic amino acid transporter gene associated with cystinuria

Biochemical Journal ◽

10.1042/bj3100951 ◽

1995 ◽

Vol 310 (3) ◽

pp. 951-955 ◽

Cited By ~ 30

Author(s):

K Miyamoto ◽

K Katai ◽

S Tatsumi ◽

K Sone ◽

H Segawa ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Regulatory Subunit ◽

Acid Transport ◽

Transport Activity ◽

Wild Type ◽

Neutral Amino Acid Transporter ◽

Acid Transporter

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

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Transport of Methylmercury through the Epithelial Type Amino Acid Transporter System B0

Journal of National Institute of Neurosciences Bangladesh ◽

10.3329/jninb.v5i2.43017 ◽

2019 ◽

Vol 5 (2) ◽

pp. 127-136

Author(s):

Rafiqul Islam ◽

Naohiko Anzai ◽

Nesar Ahmed ◽

Mohammad Ahtashamul Haque ◽

Shamima Ferdous ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Molecular Mechanism ◽

Xenopus Oocytes ◽

Critical Role ◽

Amino Acid Transporter ◽

Intestinal Lumen ◽

Dependent Manner ◽

Sodium Dependent ◽

Acid Transporter

Background: System B0 is a sodium dependent transporter that transports wide variety of neutral amino acids in the intestinal and renal proximal tubular epithelial cells. Methylmercury (MeHg) readily and non-enzymatically reacts with cysteine to form conjugate structurally similar to the amino acid methionine. Objective: In this study, we investigated the molecular mechanism of absorptive transport of MeHg in intestine using Xenopus oocytes expressing hB0AT1 and the uptake of metylmercry-Cys (MeHg-Cys) by heterodimeric amino acids transporter. Methodology: We confirmed the uptake of [14C] L-Leucine a potent substrate for the hB0AT1 amino acids transporter. The uptake of [14C] L-leucine by hB0AT1 was inhibited by MeHg-Cys conjugate, leucine, cysteine, methinine and phenylalanine in concentration–dependent manner. The IC50 of MeHg-Cys conjugate was significantly lower than that of leucine, cysteine, methinine and phenylalanine, indicating that hB0AT1 is a high affinity MeHg transporter. To assess MeHg-Cys conjugate transport, we measured [14C] MeHg uptake in Xenopus oocytes expressing hB0AT1 in presence or absence of sodium. The [14C] MeHg was transport only in the presence of cysteine and the transport was significantly sodium dependent and inhibited by a system B0 inhibitor 2-aminobicyclo-[2,21]- haptane-2-carboxylic acid (BCH). Result: The current findings indicate that hB0AT1 and heterodimeric amino acids absorb MeHg in the form of cysteine conjugate from the intestinal lumen across the brush-border membrane in to the cells and is supposed to be plays a critical role in the pathogenesis of Minamata disease and present results descried a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. Conclusion: In this experiment the results also suggest that uptake of Methionine and MeHg-Cys by heterodimeric amino acid transporter is significantly correlated where the uptake of Methionine and MeHg-Cys between heterodimeric amino acid transporter and hB0AT1 is not correlated. Journal of National Institute of Neurosciences Bangladesh, 2019;5(2): 127-136

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