Over-Production of the Human SLC7A10 in E. coli and Functional Assay in Proteoliposomes.

The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C12E8 and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na+-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.

OCTN1: A Widely Studied but Still Enigmatic Organic Cation Transporter Linked to Human Pathology and Drug Interactions.

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.

Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism.

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.

Strategies for Successful Over-Expression of Human Membrane Transport Systems Using Bacterial Hosts: Future Perspectives.

Ten percent of human genes encode for membrane transport systems, which are key components in maintaining cell homeostasis. They are involved in the transport of nutrients, catabolites, vitamins, and ions, allowing the absorption and distribution of these compounds to the various body regions. In addition, roughly 60% of FDA-approved drugs interact with membrane proteins, among which are transporters, often responsible for pharmacokinetics and side effects. Defects of membrane transport systems can cause diseases; however, knowledge of the structure/function relationships of transporters is still limited. Among the expression of hosts that produce human membrane transport systems, E. coli is one of the most favorable for its low cultivation costs, fast growth, handiness, and extensive knowledge of its genetics and molecular mechanisms. However, the expression in E. coli of human membrane proteins is often toxic due to the hydrophobicity of these proteins and the diversity in structure with respect to their bacterial counterparts. Moreover, differences in codon usage between humans and bacteria hamper translation. This review summarizes the many strategies exploited to achieve the expression of human transport systems in bacteria, providing a guide to help people who want to deal with this topic.

SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1.

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.

Chemical Approaches for Studying the Biology and Pharmacology of Membrane Transporters: The Histidine/Large Amino Acid Transporter SLC7A5 as a Benchmark.

The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.

Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4).

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 MßCD 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 MßCD, 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.

Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport.

The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent 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.

Glutamine transport. From energy supply to sensing and beyond.

Glutamine is the most abundant amino acid in plasma and is actively involved in many biosynthetic and regulatory processes. It can be synthesized endogenously but becomes "conditionally essential" in physiological or pathological conditions of high proliferation rate. To accomplish its functions glutamine has to be absorbed and distributed in the whole body. This job is efficiently carried out by a network of membrane transporters that differ in transport mechanisms and energetics, belonging to families SLC1, 6, 7, 38, and possibly, 25. Some of the transporters are involved in glutamine traffic across different membranes for metabolic purposes; others are involved in specific signaling functions through mTOR. Structure/function relationships and regulatory aspects of glutamine transporters are still at infancy. In the while, insights in involvement of these transporters in cell redox control, cancer metabolism and drug interactions are arising, stimulating basic research to uncover molecular mechanisms of transport and regulation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Acetylcholine and acetylcarnitine transport in peritoneum: Role of the SLC22A4 (OCTN1) transporter.

A suitable experimental tool based on proteoliposomes for assaying Organic Cation Transporter Novel member 1 (OCTN1) of peritoneum was pointed out. OCTN1, recently acknowledged as acetylcholine transporter, was immunodetected in rat peritoneum. Transport was assayed following flux of radiolabelled TEA, acetylcholine or acetylcarnitine in proteoliposomes reconstituted with peritoneum extract. OCTN1 mediated, besides TEA, also acetylcholine and a slower acetylcarnitine transport. External sodium inhibited acetylcholine uptake but not its release from proteoliposomes. Differently, sodium did not affect acetylcarnitine uptake. These results suggested that physiologically, acetylcholine should be released while acetylcarnitine was taken up by peritoneum cells. Transport was impaired by OCTN1 inhibitors, butyrobetaine, spermine, and choline. Biotin was also found as acetylcholine transport inhibitor. Anti-OCTN1 antibody specifically inhibited acetylcholine transport confirming the involvement of OCTN1. The transporter was also immunodetected in human mesothelial primary cells. Extract from these cells was reconstituted in proteoliposomes. Transport features very similar to those found with rat peritoneum were observed. Validation of the proteoliposome model for peritoneal transport study was then achieved assaying transport in intact mesothelial cells. TEA, butyrobetaine and Na(+) inhibited acetylcholine transport in intact cells while efflux was Na(+) insensitive. Therefore transport features in intact cells overlapped those found in proteoliposomes.

Bacterial production and reconstitution in proteoliposomes of Solanum lycopersicum CAT2: a transporter of basic amino acids and organic cations.

KEY MESSAGE: The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes. Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cations Tetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 in signaling. In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [3H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [3H]Ornithine and [3H]Lys was also detected at lower efficiency with respect to [3H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na(+). Internal Na(+) slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [(3)H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [(3)H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na(+) on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.

Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5).

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na(+)-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K(+) gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na(+). Internal Na(+) exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.

Inflammation and Organic Cation Transporters Novel (OCTNs).

Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.

OCTN1 (SLC22A4) displays two different transport pathways for organic cations or zwitterions.

BACKGROUND: OCTN1 belongs to the SLC22 family, which includes transporters for cationic, zwitterionic, and anionic substrates. OCTN1 function and role in cells are still poorly understood. Not only cations, such as TEA, but also zwitterions, such as carnitine and ergothioneine, figure among transported molecules. METHODS: In this work, we carried out transport assays measuring [14C]-TEA and [3H]-Carnitine in proteoliposomes reconstituted with the recombinant human OCTN1 in the presence of Na+ or other cations. The homology model of OCTN1 was built using the structure of OCT3 as a template for docking analysis. RESULTS: TEA and carnitine did not inhibit each other. Moreover, carnitine uptake was not affected by the presence of Na+ and TEBA, whereas TEA was strongly inhibited by both compounds. Computational data revealed that TEA, Na+, and carnitine can interact with E381 in the OCTN1 substrate site. Differently from TEA, in the presence of Na+, carnitine is still able to interact with the binding site via R469. CONCLUSIONS: The lack of mutual inhibition of the two prototype substrates, the different effect of Na+ and TEBA on their transport reaction, together with the computational analysis supports the existence of two transport pathways for cations and zwitterions. GENERAL SIGNIFICANCE: The results shed new light on the transport mechanisms of OCTN1, helping to get further insights into the structure/function relationships. The described results correlate well with previous and very recent findings on the polyspecificity of the OCT group of transporters belonging to the same family.

Atomistic description of the OCTN1 recognition mechanism via in silico methods.

The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease.

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [(3)H]acetylcholine, the Km of transport being 1.0mM with V(max) of 160nmol⋅mg(-1)protein⋅min(-1). OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [(3)H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V(max) was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohn's disease-associated 503F variant, revealed time course, Km and V(max) for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V(max) of OCTN1 503F-mediated acetylcholine efflux (1.9nmol⋅mg(-1)protein⋅min(-1)) was significantly lower than that of wild-type OCTN1 (14nmol⋅mg(-1)protein⋅min(-1)). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohn's disease.

Inhibition of the OCTN2 carnitine transporter by HgCl2 and methylmercury in the proteoliposome experimental model: insights in the mechanism of toxicity.

Mercury causes toxic effects in many tissues interacting with protein cysteine (Cys) thiols. Transport systems represent critical targets of mercurials. Indeed, the majority of transport systems of higher eukaryotes contain several Cys residues. One of the most up to date method of studying transport is the reconstitution of transporters in proteoliposomes. This method has been used as a useful approach to test the effect of HgCl(2) and methylmercury (MeHg) on the carnitine (OCTN2) transporter. OCTN2, extracted from kidney brush border membranes with 3% octaethylene glycol monododecyl ether (C(12)E(8)), was reconstituted in liposomes by removing the detergent with hydrophobic chromatography columns. Transport was measured as [(3)H]carnitine uptake into proteoliposomes containing carnitine (antiport reaction). Mercurials strongly inhibited the antiport. Inhibition was reversed by 1,4-dithioerythritol, L-cysteine (Cys), and N-acetyl-L-cysteine (NAC) indicating that it was caused by covalent reaction of mercurials with Cys residue(s). The IC(50) for HgCl(2), and MeHg were 2.5 and 7.4 µM, respectively. Kinetic studies showed non competitive or mixed inhibition for HgCl(2) or MeHg with Ki of 4.2 and 13 µM, respectively. The presence of substrate prevented the inhibition indicating that the mercurial binding residue (Cys) is in the substrate binding site. Efflux of carnitine from proteoliposomes was trans-stimulated, not inhibited, by higher concentrations (500 µM) of extraliposomal MeHg and HgCl(2). Differently, no effects on uptake of carnitine were exerted by mercurials present in the internal compartment of the proteoliposomes. The results allowed gaining new insights in the molecular mechanism of inhibition and of mercurial toxicity.

The B°AT1 amino acid transporter from rat kidney reconstituted in liposomes: kinetics and inactivation by methylmercury.

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicating that it was not required for the transport function. Neutral amino acids behaved as competitive inhibitors of the glutamine transport mediated by B°AT1 with half saturation constants ranging from 0.13 to 4.74mM. The intraliposomal half saturation constant for glutamine was 2.0mM. By a bisubstrate kinetic analysis of the glutamine-Na(+) cotransport, a random simultaneous mechanism was found. Methylmercury and HgCl(2) inhibited the transporter; the inhibition was reversed by dithioerythritol, Cys and, at a lower extent, N-acetylcysteine but not by S-carboxymethylcysteine. The IC(50) of the transporter for methylmercury and HgCl(2) was 1.88 and 1.75µM, respectively. The reagents behaved as non-competitive inhibitors toward both glutamine and Na(+) and no protection by glutamine or Na(+) was found for the two inhibitors.

Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: the mouse carnitine transporter OCTN3.

pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni(2+)-chelating chromatography with a yield of 2mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [(3)H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K(m) for carnitine was 36 µM. [(3)H]carnitine transport was inhibited by carnitine analogues and some Cys and NH(2) reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.