The Mutation F227I Increases the Coupling of Metal Ion Transport in DCT1

Abstract

Metal ion transport by DCT1, a member of the natural resistance-associated macrophage protein family, is driven by protons. The stoichiometry of the proton to metal ion is variable, and under optimal transport conditions, more than 10 protons are co-transported with a single metal ion. To understand this phenomenon better, we used site-directed mutagenesis of DCT1 and analyzed the mutants by complementation of yeast suppressor of mitochondria import function-null mutants and electrophysiology with Xenopus oocytes. The mutation F227I resulted in an increase of up to 14-fold in the ratio between metal ions to protons transported. This observation suggests that low metal ion to proton transport of DCT1 resulting from a proton slippage is not a necessity of the transport mechanism in which positively charged protons are driving two positive charges of the metal ion in the same direction. It supports the idea that the proton slippage has a physiological advantage, and the proton slip was positively selected during the evolution of DCT1.Metal ions are vital elements for all living cells. The NRAMP 1 family of metal ion transporters apparently plays a major role in metal ion homeostasis (1-4). Most of the information available about the mechanism of these transporters has resulted from studies of the family members NRAMP2 (DCT1) from mammalian and SMF1 from yeast expressed in Xenopus oocyte (5-9). Xenopus oocytes have a very low metal ion uptake background, which makes them the ideal heterologous expression system for metal ion transporters. In addition to the uptake measurements of the various divalent cations, it is possible to analyze the electrophysiological parameters generated by imposed potentials in the transporter-expressing oocyte as compared with control (10). The studies of DCT1 and Smf1p demonstrated that both of them function as general divalent metal ion transporters and that a proton gradient is the driving force for the metal ion transport (8, 9). However, the transport of protons and metal ions is "loosely coupled" because the process exhibits a variable stoichiometry. At pH 7 and membrane potentials of Ϫ90 to Ϫ30 mV, DCT1 transports one Fe 2ϩ ion with one H ϩ . At high proton concentration (low pH), the number of H ϩ ions transported with one Fe 2ϩ ion increased to 10 (8). Moreover, on changing the membrane potential from ϩ10 to Ϫ80 mV at this low pH, the number of H ϩ ions transported with one Fe 2ϩ ion increased from 3 to ϳ18 (9). In DCT1, this phenomenon was defined as a metal ion-dependent proton slippage (1, 9, 11). In contrast to DCT1, Smf1p showed a metal ion-independent sodium slip through the proton-translocating pathway (9, 12). The mechanism of this phenomenon is not well characterized, and the sites on the transporters that generate it are not known.In this work, we addressed the coupling between proton and metal ion transport, and report on a mutation in DCT1 that exhibits an ϳ14-fold increase in the ratio of metal ion to proton transport. MATERIALS AND METHODSSite-directed Mutagenesis o...

Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import

“…S1, F-H). Overall, these trends are similar to the voltage dependence of mammalian Nramp2 Fe 2+ transport observed via electrophysiology (Gunshin et al, 1997;Chen et al, 1999;Nevo and Nelson, 2004;Xu et al, 2004;Mackenzie et al, 2006Mackenzie et al, , 2007Pujol-Giménez et al, 2017) and transport assays using radioactive Fe 2+ (Chen et al, 1999).…”

Divalent metal transporter 1 is involved in amyloid precursor protein processing and Aβ generation

“…We have recently reported that several members of a zinc trans-porter (ZNT) family are abundantly expressed in APP/ PS1 mouse brain (17). Together, with the present data, we provide the hypothesis that involvement of zinc in regulation of APP processing may be more related to the ZNT family rather than DMT1, although zinc transmembrane transportation is regulated by DMT1 (27). For ferric iron, it has been reported that DMT1 transports divalent (ferrous) iron, most likely produced by the reduction of ferric iron at the cell surface or within endosomal compartments (55), although there may be a separate pathway for ferric iron that does not rely on DMT1 (56).…”

“…Divalent metal transporter 1 (DMT1), also known as natural resistance-associated macrophage protein 2 (Nramp2) or divalent cation transporter 1 (DCT1), is a newly discovered proton-coupled metal-ion transport protein: the first mammalian transmembrane iron transporter (22). A widely expressed protein with 12 putative transmembrane-spanning domains, DMT1 is responsible for the uptake of a broad range of divalent metal ions, including Fe 2ϩ , Zn 2ϩ , Mn 2ϩ , Co 2ϩ , Cd 2ϩ , Cu 2ϩ , Ni 2ϩ , and Pb 2ϩ (22)(23)(24)(25)(26)(27). Four isoforms of DMT1 protein are distinguished through their variant mRNA transcripts that vary both at their 5Ј-UTR and 3Ј-UTR.…”

Identification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium

“…Several studies have investigated the structure–function relationships in proteins of the NRAMP family (Lam‐Yuk‐Tseung et al ., ; Nevo and Nelson, , ; Courville et al ., ). Interestingly, analysis of the G185R mutation in the fourth transmembrane domain of NRAMP2/DCT1/DMT1, which leads to microcytic anemia in rat and mouse, demonstrated that this mutation not only impairs Fe transport but converts the transporter into a Ca‐permeable channel (Xu et al ., ).…”