title: Functional and molecular effects of mercury compounds on the human OCTN1 cation transporter: C50 and C136 are the targets for potent inhibition.
abstract: The effect of mercury compounds has been tested on the organic cation transporter, hOCTN1. MeHg(+), Hg(2+), or Cd(2+) caused strong inhibition of transport. 1,4-Dithioerythritol (DTE), cysteine (Cys), and N-acetyl-l-cysteine reversed (NAC) the inhibition at different extents. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), a prototype SH reagent, exerted inhibition of transport similar to that observed for the mercurial agents. To investigate the mechanism of action of mercurials, mutants of hOCTN1 in which each of the Cys residues was substituted by Ala have been constructed, over-expressed in Escherichia coli, and purified. Tetraethylammonium chloride (TEA) uptake mediated by each mutant in proteoliposomes was comparable to that of wild type (WT). IC50 values of the WT and mutants for the mercury compounds were derived from dose-response analyses. The mutants C50A and C136A showed significant increase of IC50 indicating that the 2 Cys residues were involved in the interaction with the mercury compounds and inhibition of the transporter. The double mutant C50A/C136A was constructed; the lack of inhibition confirmed that the 2 Cys residues are the targets of mercury compounds. MTSEA showed similar behavior with respect to the mercurial reagents with the difference that increased IC50 was observed also in the C81A mutant. Similar results were obtained when transport was measured as acetylcholine uptake. Ethyl mercury (Thimerosal) inhibited hOCTN1 as well. C50A, C50A/C136A and, at very lower extent, C136A showed increased IC50 indicating that C50 was the major target of this mercury compound. The homology model of hOCTN1 was built using as template PiPT and validated by the experimental data on mutant proteins.
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Galluccio
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Pochini
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Peta
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Iannì
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy.
authors.forename: Mariafrancesca
authors.initials: M
authors.lastname: Scalise
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, Via Bucci 4C, University of Calabria, 87036 Arcavacata di Rende, Italy cesare.indiveri@unical.it.
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D001692:Biological Transport; D019256:Cadmium Chloride / Q000633:toxicity; D003545:Cysteine; D004305:Dose-Response Relationship, Drug; D005838:Genotype; D006801:Humans; D007700:Kinetics; D008627:Mercuric Chloride / Q000633:toxicity*; D008767:Methylmercury Compounds / Q000633:toxicity*; D008958:Models, Molecular; D009154:Mutation; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship; D027981:Symporters; D019789:Tetraethylammonium / Q000378:metabolism; D013849:Thimerosal / Q000633:toxicity*
chemical_list: D008767:Methylmercury Compounds; D027701:Organic Cation Transport Proteins; D011994:Recombinant Proteins; C109807:SLC22A4 protein, human; D027981:Symporters; D013849:Thimerosal; D008627:Mercuric Chloride; D019789:Tetraethylammonium; D019256:Cadmium Chloride; D003545:Cysteine; D000109:Acetylcholine; C004925:methylmercuric chloride
keywords: acetylcholine; ethylmercury; liposomes; methylmercury; mutagenesis; toxicology