Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1.
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.
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
Anthony C
AC
Illing
Ali
A
Shawki
Christopher L
CL
Cunningham
Bryan
B
Mackenzie
D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes; D009930:Organic Chemicals / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity / Q000502:physiology; D014982:Xenopus laevis; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D002412:Cations; D019216:Metals, Heavy; D009930:Organic Chemicals; D000099236:Solute Carrier Family 11, Member 2; C431805:Phen Green SK