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 HgCl2 and methylmercury (MeHg) on the carnitine (OCTN2) transporter. OCTN2, extracted from kidney brush border membranes with 3% octaethylene glycol monododecyl ether (C12E8), was reconstituted in liposomes by removing the detergent with hydrophobic chromatography columns. Transport was measured as [H-3]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 IC50 for HgCl2, and MeHg were 2.5 and 7.4 mu M, respectively. Kinetic studies showed non competitive or mixed inhibition for HgCl2 or MeHg with Ki of 4.2 and 13 mu M, 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 mu M) of extraliposomal MeHg and HgCl2. 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.