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Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1.

ABSTRACT: Divalent metal-ion transporter-1 (DMT1) is a H(+)-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio I(max)/K(0.5) (determined from evoked currents at -70 mV): Cd(2+) > Fe(2+) > Co(2+), Mn(2+) ? Zn(2+), Ni(2+), VO(2+). DMT1 expression did not stimulate the transport of Cr(2+), Cr(3+), Cu(+), Cu(2+), Fe(3+), Ga(3+), Hg(2+), or VO(+). (55)Fe(2+) transport was competitively inhibited by Co(2+) and Mn(2+). Zn(2+) only weakly inhibited (55)Fe(2+) transport. Our data reveal that DMT1 selects Fe(2+) over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported.

SUBMITTER: Illing AC

PROVIDER: S-EPMC3436370 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1.

Illing Anthony C AC Shawki Ali A Cunningham Christopher L CL Mackenzie Bryan B

The Journal of biological chemistry 20120626 36

Divalent metal-ion transporter-1 (DMT1) is a H(+)-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluoresc ...[more]

PMID: 22736759

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Project description:DMT1 (divalent metal-ion transporter 1) is a widely expressed metal-ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body, including erythroid precursors. Mutations in DMT1 cause severe microcytic anaemia in animal models. Four DMT1 isoforms that differ in their N- and C-termini arise from mRNA transcripts that vary both at their 5'-ends (starting in exon 1A or exon 1B) and at their 3'-ends giving rise to mRNAs containing (+) or lacking (-) the 3'-IRE (iron-responsive element) and resulting in altered C-terminal coding sequences. To determine whether these variations result in functional differences between isoforms, we explored the functional properties of each isoform using the voltage clamp and radiotracer assays in cRNA-injected Xenopus oocytes. 1A/IRE+-DMT1 mediated Fe2+-evoked currents that were saturable (K(0.5)(Fe) approximately 1-2 microM), temperature-dependent (Q10 approximately 2), H+-dependent (K(0.5)(H) approximately 1 muM) and voltage-dependent. 1A/IRE+-DMT1 exhibited the provisional substrate profile (ranked on currents) Cd2+, Co2+, Fe2+, Mn2+>Ni2+, V3+>>Pb2+. Zn2+ also evoked large currents; however, the zinc-evoked current was accounted for by H+ and Cl- conductances and was not associated with significant Zn2+ transport. 1B/IRE+-DMT1 exhibited the same substrate profile, Fe2+ affinity and dependence on the H+ electrochemical gradient. Each isoform mediated 55Fe2+ uptake and Fe2+-evoked currents at low extracellular pH. Whereas iron transport activity varied markedly between the four isoforms, the activity for each correlated with the density of anti-DMT1 immunostaining in the plasma membrane, and the turnover rate of the Fe2+ transport cycle did not differ between isoforms. Therefore all four isoforms of human DMT1 function as metal-ion transporters of equivalent efficiency. Our results reveal that the N- and C-terminal sequence variations among the DMT1 isoforms do not alter DMT1 functional properties. We therefore propose that these variations serve as tissue-specific signals or cues to direct DMT1 to the appropriate subcellular compartments (e.g. in erythroid cells) or the plasma membrane (e.g. in intestine).

Project description:NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-d-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogues. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apoenzyme reactivated upon incubation with 1 equiv of Zn2+, Cd2+, Co2+, Mn2+, Ni2+, or Fe2+. In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate (Asp-273) found at the end of beta-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D2O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton-transfer steps or the release of products. N-Trifluoroacetyl-d-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.

Dataset Information

Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1.

Publications

Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1.

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