title: Mitochondrial carnitine/acylcarnitine transporter, a novel target of mercury toxicity.
abstract: The effect of Hg(2+) and CH3Hg(+) on the mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied on the recombinant protein and on the CACT extracted from HeLa cells or Zebrafish and reconstituted in proteoliposomes. Transport was abolished upon treatment of the recombinant CACT in proteoliposomes by Hg(2+) or CH3Hg(+). Inhibition was reversed by the SH reducing agent 1,4-dithioerythritol, GSH, and N-acetylcysteine. IC50 for Hg(2+) and CH3Hg(+) of 90 nM and 137 nM, respectively, were measured by dose-response analyses. Inhibition was abolished in the C-less CACT mutant. Strong reduction of inhibition by both reagents was observed in the C136A and some reduction in the C155A mutants. Inhibition similar to that of the WT was observed in the C23V/C58V/C89S/C155V/C283S mutant, containing only C136. Optimal inhibition by Hg(2+)was found in the four replacement mutants C23V/C58V/C89S/C283S containing both C136 and C155 indicating cross-reaction of Hg(2+) with the two Cys residues. Inhibition kinetic analysis showed mixed inhibition by Hg(2+) or competitive inhibition by CH3Hg(+). HeLa cells or Zebrafish were treated with the more potent inhibitor. Ten micromolar HgCl2 caused clear impairment of viability of HeLa cells. The transport assay in proteoliposomes with CACT extracted from treated cells showed that the transporter was inactivated and that DTE rescued the activity. Nearly identical results were observed with Zebrafish upon extraction of the CACT from the liver of the treated animals that, indeed, showed accumulation of the mercurial compound.
authors: †CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy.|‡Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.
authors: Annamaria
authors: A
authors: Tonazzi
authors: †CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy.|‡Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.
authors: Nicola
authors: N
authors: Giangregorio
authors: §Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Lara
authors: L
authors: Console
authors: §Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Mariafrancesca
authors: M
authors: Scalise
authors: §Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Daniele
authors: D
authors: La Russa
authors: ‡Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.
authors: Caterina
authors: C
authors: Notaristefano
authors: §Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Elvira
authors: E
authors: Brunelli
authors: §Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Donatella
authors: D
authors: Barca
authors: †CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy.|§Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors: Cesare
authors: C
authors: Indiveri
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008628:Mercury / Q000633:toxicity*; D008767:Methylmercury Compounds / Q000633:toxicity*; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D026901:Membrane Transport Proteins; D008767:Methylmercury Compounds; D033681:Mitochondrial Membrane Transport Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C020485:proteoliposomes; C120109:SLC25A20 protein, human; D008628:Mercury