Effects of heavy metal cations on the mitochondrial ornithine/citrulline transporter reconstituted in liposomes

The effect of heavy metal cations on the mitochondrial ornithine/citrulline transporter was tested in proteoliposomes reconstituted with the protein purified from rat liver. The transport activity was measured as [³H]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [³H]ornithine efflux in the absence of external substrate (ornithine/H⁺ transport mode). 0.1 mM Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺ and Zn²⁺ strongly inhibited (more than 85%) the antiport; whereas Mn²⁺, Co²⁺ and Ni²⁺ inhibited less efficiently (25, 47 and 69%, respectively). The IC₅₀ values of the transporter for the different metal ions ranged from 0.71 to 350 μM. Co²⁺ and Ni²⁺ also inhibited the [³H]ornithine efflux whereas Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺ and Zn²⁺ stimulated the [³H]ornithine efflux. The stimulation of the [³H]ornithine efflux by Cu²⁺ and Cd²⁺ (as well as by Pb²⁺, Hg²⁺ and Zn²⁺) was not prevented by NEM and was reversed by DTE. These features indicated that the inhibition of the antiport was due to the interaction of the Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺ and Zn²⁺ with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas the stimulation of [³H]ornithine efflux was due to the induction of a pore-like function of the transporter caused by interaction of cations with a different population of SH groups. Differently, the inhibition of the ornithine transporter by Ni²⁺, Co²⁺ or Mn²⁺ was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition.