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The plasma membrane transporter ASCT2 is a well-known Na+-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh. [ABSTRACT FROM AUTHOR]

Alanine, serine, cysteine transporter 2 (ASCT2) is a membrane amino acid transporter with relevance to human physiology and pathology, such as cancer. Notwithstanding, the study on the ASCT2 transport cycle still has unknown aspects, such as the role of Na+ in this process. We investigate this issue using recombinant hASCT2 reconstituted in proteoliposomes. Changes in the composition of purification buffers show the crucial role of Na+ in ASCT2 functionality. The transport activity is abolished when Na+ is absent or substituted by Li+ or K+ in purification buffers. By employing a Na+ fluorometric probe, we measured an inwardly directed flux of Na+ and, by combining fluorometric and radiometric assays, determined a 2Na+ : 1Gln stoichiometry. Kinetics of Na+ transport suggest that pH‐sensitive residues are involved in Na+ binding/transport. Our results clarify the role of Na+ on human ASCT2 transporter activity. [ABSTRACT FROM AUTHOR]

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1. [ABSTRACT FROM AUTHOR]

Objective The study aimed to evaluate whether high-flow oxygen therapy (HFOT) during training was more effective than oxygen in improving exercise capacity in hypoxemic chronic obstructive pulmonary disease (COPD). Methods A total of 171 patients with COPD and chronic hypoxemia were consecutively recruited in 8 rehabilitation hospitals in a randomized controlled trial. Cycle-ergometer exercise training was used in 20 supervised sessions at iso inspiratory oxygen fraction in both groups. Pre- and post-training endurance time (Tlim), 6-minute walking distance (6MWD), respiratory and limb muscle strength, arterial blood gases, Barthel Index, Barthel Dyspnea Index, COPD Assessment Test, Maugeri Respiratory Failure questionnaire, and patient satisfaction were evaluated. Results Due to 15.4% and 24.1% dropout rates, 71 and 66 patients were analyzed in HFOT and Venturi mask (V-mask) groups, respectively. Exercise capacity significantly improved after training in both groups with similar patient satisfaction. Between-group difference in post-training improvement in 6MWD (mean: 17.14 m; 95% CI = 0.87 to 33.43 m) but not in Tlim (mean: 141.85 seconds; 95% CI = −18.72 to 302.42 seconds) was significantly higher in HFOT. The minimal clinically important difference of Tlim was reached by 47% of patients in the V-mask group and 56% of patients in the HFOT group, whereas the minimal clinically important difference of 6MWD was reached by 51% of patients in the V-mask group and 69% of patients in the HFOT group, respectively. Conclusion In patients with hypoxemic COPD, exercise training is effective in improving exercise capacity. Impact Statement The addition of HFOT during exercise training is not more effective than oxygen through V-mask in improving endurance time, the primary outcome, whereas it is more effective in improving walking distance. [ABSTRACT FROM AUTHOR]

The human plasma membrane transporter ASCT2 is responsible for mediating Nadependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2. [ABSTRACT FROM AUTHOR]

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 µM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-3,4-dIpyrimidine (PP-2) abolished the effect. A concentration of 50 µM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment. [ABSTRACT FROM AUTHOR]

The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology. [ABSTRACT FROM AUTHOR]