Mitochondrial Carnitine/Acylcarnitine Transporter, a Novel Target of Mercury Toxicity

The effect of Hg²⁺ and CH<inf>3</inf>Hg⁺ 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 Hg²⁺ or CH<inf>3</inf>Hg⁺. Inhibition was reversed by the SH reducing agent 1,4-dithioerythritol, GSH, and N-acetylcysteine. IC<inf>50</inf> for Hg²⁺ and CH<inf>3</inf>Hg⁺ 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 Hg²⁺was found in the four replacement mutants C23V/C58V/C89S/C283S containing both C136 and C155 indicating cross-reaction of Hg²⁺ with the two Cys residues. Inhibition kinetic analysis showed mixed inhibition by Hg²⁺ or competitive inhibition by CH<inf>3</inf>Hg⁺. HeLa cells or Zebrafish were treated with the more potent inhibitor. Ten micromolar HgCl<inf>2</inf> 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. (Chemical Presented).