Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in <i>Xenopus laevis</i> oocytes

Abstract

Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life. Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells. Little is known about the transport mechanism of metal ions by eukaryotic cells. Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters. Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a 'slippage' phenomenon of different monovalent cations unique to each transporter. Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties. We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to 'slip' through the DCT1 transporter. A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells. 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials. In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials. Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered. Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential. A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed. The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed.