Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

Mackenzie, Bryan; Ujwal, M. L.; Chang, Min; Romero, Michael F.; Hediger, Matthias A.

doi:10.1007/s00424-005-1494-3

Cited by 132 publications

(207 citation statements)

References 58 publications

(81 reference statements)

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“…These were isolated from capacitive transient currents (which decayed with half-times of 0.3-0.9 ms) and steady-state currents by the fitted method (9,15,21,22). The compensated currents thus obtained were integrated with time to obtain charge movement (Q).…”

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

“…We used L-[1-14 C]ascorbic acid at final specific activity 0.3-3 GBq/mmol and 22 Na at a final specific activity 0.34 MBq/mg (both from Perkin-Elmer Life Sciences). At the end of the 5-min uptake period, oocytes were rinsed with ice-cold ChoCl medium and solubilized with 5% SDS before 14 C or 22 Na content was assayed by liquid scintillation counting.…”

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

“…We determined Na ϩ -L-ascorbic acid coupling stoichiometry of SVCT1 by measuring 22 Na ϩ and L-[ 14 C]ascorbic acid fluxes in individual oocytes under voltage clamp and relating the fluxes to net charge translocation (Fig. 2).…”

Section: Currents Associated With Expression Of Human Svct1 Inmentioning

confidence: 99%

“…L-Ascorbic acid-evoked currents were integrated with time to obtain charge and converted to a molar equivalent (Q Asc in pmol) using the Faraday constant (9.65 ϫ 10 4 C/mol). A: typical current record for an individual oocyte superfused initially with 50 mM unlabeled Na ϩ and then 50 mM 22 Na ϩ plus 1 mM L-ascorbic acid (L-Asc) for the period shown by the filled bar. L-Ascorbic acid-dependent charge (Q Asc ) was calculated as the gray shaded area.…”

Section: Currents Associated With Expression Of Human Svct1 Inmentioning

confidence: 99%

“…Here we explored its molecular mechanisms by testing the hypothesis that the Na ϩ dependence and rheogenicity of SVCT1 arise from coupled transport of two Na ϩ and one L-ascorbic acid per transport cycle and that L-ascorbic acid transport is driven by the electrochemical gradient for Na ϩ . Our approach was to measure radiotracer (L-[ 14 C]ascorbic acid, 22 Na) fluxes and currents in Xenopus oocytes expressing SVCT1 and to model the kinetic mechanisms of SVCT1 with the aid of computer simulation.…”

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Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mackenzie¹,

Illing²,

Hediger³

2008

American Journal of Physiology-Cell Physiology

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Mackenzie B, Illing AC, Hediger MA. Transport model of the human Na ϩ -coupled L-ascorbic acid (vitamin C) transporter SVCT1. Am J Physiol Cell Physiol 294: C451-C459, 2008. First published December 19, 2007 doi:10.1152/ajpcell.00439.2007.-Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-Ascorbic acid transport was saturable (K0.5 Ϸ 70 M), temperature dependent (Q10 Ϸ 5), and energized by the Na ϩ electrochemical potential gradient. We obtained a Na ϩ -L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-Ascorbic acid and Na ϩ saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na ϩ , L-ascorbic acid, Na ϩ . In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na ϩ , maximal charge translocation (Qmax) was Ϸ25 nC, around a midpoint (V0.5) at Ϫ9 mV, and with apparent valence ϷϪ1. Qmax was conserved upon progressive removal of Na ϩ , whereas V0.5 shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na ϩ partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease. cotransporters; sodium dependent; intestinal absorption; model simulation; renal reabsorption; Xenopus oocyte VITAMIN C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant scavenger of free radicals and as a cofactor in many enzymatic reactions (3,6,10,26,29). It cannot be synthesized in Homo sapiens and must be derived from the diet. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial Na ϩ -dependent Lascorbic acid cotransporter SVCT1 (reviewed in Refs. 32 and 35), the product of the SLC23A1 gene. Selective sorting of SVCT1 protein to the apical membrane has been demonstrated in cultured cell lines (Caco2 and MDCK) of intestinal and renal origin (2, 23).We and others previously demonstrated that human SVCT1 is a Na ϩ -dependent transporter that favors L-ascorbic acid over D-isoascorbic acid and that the oxidized form of vitamin C, dehydroascorbic acid, is excluded (5, 34). Here we explored its molecular mechanisms by testing the hypothesis that the Na ϩ dependence and rheogenicity of SVCT1 arise from...

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Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

Section: Currents Associated With Expression Of Human Svct1 Inmentioning

confidence: 99%

Section: Currents Associated With Expression Of Human Svct1 Inmentioning

confidence: 99%

mentioning

confidence: 99%

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Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mackenzie¹,

Illing²,

Hediger³

2008

American Journal of Physiology-Cell Physiology

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Mammalian Iron Metabolism and Dietary Iron Absorption

Crichton

2016

Iron Metabolism

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Ion channel activity of transmembrane segment 6 of Escherichia coli proton‐dependent manganese transporter

et al. 2010

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Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure-function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His(211) were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure-function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein.

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Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

Cited by 132 publications

References 58 publications

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mammalian Iron Metabolism and Dietary Iron Absorption

Ion channel activity of transmembrane segment 6 of Escherichia coli proton‐dependent manganese transporter

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Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

Cited by 132 publications

References 58 publications

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mammalian Iron Metabolism and Dietary Iron Absorption

Ion channel activity of transmembrane segment 6 of Escherichia coli proton‐dependent manganese transporter

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Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1