scholarly journals B0AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Function ◽  
2021 ◽  
Author(s):  
Bruce R Stevens ◽  
J Clive Ellory ◽  
Robert L Preston
Keyword(s):  
Amino Acid ◽  
Membrane Trafficking ◽  
Functional Unit ◽  
Membrane Vesicles ◽  
High Energy ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Transport Activity ◽  
Acid Transporter

Abstract The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B0AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).

scholarly journals Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays

2018 ◽  
Vol 24 (2) ◽  
pp. 111-120 ◽  
Author(s):  
Sanjay J. Danthi ◽  
Beirong Liang ◽  
Oanh Smicker ◽  
Benjamin Coupland ◽  
Jill Gregory ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput Screening ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Imaging Plate ◽  
Acid Uptake ◽  
Functional Assays ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter ◽  
Pharmacologically Active

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

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+.

The SLC1 high-affinity glutamate and neutral amino acid transporter family

2013 ◽  
Vol 34 (2-3) ◽  
pp. 108-120 ◽  
Author(s):  
Yoshikatsu Kanai ◽  
Benjamin Clémençon ◽  
Alexandre Simonin ◽  
Michele Leuenberger ◽  
Martin Lochner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
High Affinity ◽  
Neutral Amino Acid Transporter ◽  
Transporter Family ◽  
Acid Transporter

scholarly journals Characterization of the amino acid response element within the human sodium-coupled neutral amino acid transporter 2 (SNAT2) System A transporter gene

2006 ◽  
Vol 395 (3) ◽  
pp. 517-527 ◽  
Author(s):  
Stela S. Palii ◽  
Michelle M. Thiaville ◽  
Yuan-Xiang Pan ◽  
Can Zhong ◽  
Michael S. Kilberg
Keyword(s):  
Amino Acid ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
System A ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter ◽  
Amino Acid Response

The neutral amino acid transport activity, System A, is enhanced by amino acid limitation of mammalian cells. Of the three gene products that encode System A activity, the one that exhibits this regulation is SNAT2 (sodium-coupled neutral amino acid transporter 2). Fibroblasts that are deficient in the amino acid response pathway exhibited little or no induction of SNAT2 mRNA. Synthesis of SNAT2 mRNA increased within 1–2 h after amino acid removal from HepG2 human hepatoma cells. The amino acid responsive SNAT2 genomic element that mediates the regulation has been localized to the first intron. Increased binding of selected members of the ATF (activating transcription factor) and C/EBP (CCAAT/enhancer-binding protein) families to the intronic enhancer was established both in vitro and in vivo. In contrast, there was no significant association of these factors with the SNAT2 promoter. Expression of exogenous individual ATF and C/EBP proteins documented that specific family members are associated with either activation or repression of SNAT2 transcription. Chromatin immunoprecipitation analysis established in vivo that amino acid deprivation led to increased RNA polymerase II recruitment to the SNAT2 promoter.

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