Divalent metal-ion transporter DMT1 and its role in intestinal metal-ion uptake

描述（由申请人提供）：DMT 1是一种广泛表达的铁转运蛋白，对于铁的充分肠道吸收和红细胞前体中铁的转运以产生血红蛋白至关重要。DMT 1的罕见突变导致严重的小细胞性贫血。相反，遗传性血色病（白种人中最常见的遗传性疾病）中DMT 1的失调导致重要器官中的毒性铁过载。由于不存在其排泄的调节机制，铁稳态通过调节肠铁吸收来实现。DMT 1是铁吸收的通道-它是非血红素铁的主要或唯一的顶端转运蛋白-使其成为本提案的焦点。缺铁影响多达10%的美国人口，是世界上最普遍的微量营养素缺乏症。铁缺乏是镉中毒的一个严重危险因素，这表明镉和铁有着共同的吸收机制。在这项建议的前提下，DMT 1是一个复杂的H+耦合和电压依赖性亚铁（Fe 2+）转运蛋白，并在肠道刷状缘膜的酸性微气候提供了质子动力激励DMT 1的调查。我们将探讨H+耦合的分子基础，并探讨通过DMT 1使用电压钳，放射性示踪剂（55 Fe）测定，并在非洲爪蟾卵母细胞表达野生型DMT 1或突变蛋白的荧光方法显着的非耦合H+通量（滑移）的生理作用。接下来，我们将使用放射性示踪剂和荧光方法研究小鼠肠道中的铁转运;使用肠Na+/H+交换器的特异性抑制剂（NHE 3和NHE 2）和缺乏NHE 3，NHE 2，或者胃H+/K+-ATP酶将允许我们评估胃酸和肠刷状缘酸性微气候在提供H+驱动DMT 1介导的Fe 2+中的作用运输第二个前提是，DMT 1不仅吸收铁，还吸收某些其他必需金属，如Co和Mn（以及微量金属，如Ni和V），但这种混杂性也使其成为有毒重金属Cd的主要进入途径。在这里，我们将使用我们的卵母细胞测定来确定DMT 1的全面底物概况和金属离子选择性。接下来，我们将使用贝尔格莱德贫血大鼠（其在DMT 1中具有突变）来检查DMT 1在其能够运输的每种金属的吸收中的生理意义。最后，我们将确定DMT 1中的金属配位位点。以这种方式研究DMT 1将导致更好地理解非血红素铁吸收的机制，有效吸收所需的条件以及DMT 1在其他过渡金属代谢中的作用。这项工作的结果将有助于推动改善金属营养或治疗铁过载和重金属中毒的新策略的发展。 公共卫生相关性：缺铁会导致贫血，遗传性疾病血色病会导致毒性铁过载，说明平衡肠道铁吸收的重要性。膳食铁进入肠道细胞的通道是一种名为DMT 1的蛋白质，它也可以运输有毒金属镉。在这个项目中，我们将在分子水平上研究铁和镉通过DMT 1的转运，以推动新策略的发展，以改善铁营养和防止镉或铁过载的毒性作用。

DESCRIPTION (provided by applicant): DMT1 is a widely-expressed iron transporter that is essential for adequate intestinal absorption of iron and for transport of iron in red blood cell precursors for the production of hemoglobin. Rare mutations in DMT1 cause severe microcytic anemia. Conversely, dysregulation of DMT1 in hereditary hemochromatosis, the most common hereditary disease in Caucasians, results in toxic iron overload in vital organs. Since there exists no regulated mechanism for its excretion, iron homeostasis is achieved by regulating intestinal iron absorption. DMT1 is the gateway for iron absorption - it is the primary or only apical transporter of nonheme iron - making it the focus of this proposal. Iron deficiency affects as much as 10% of the U.S. population and is the most prevalent micronutrient deficiency worldwide. Important for this proposal, iron deficiency is a serious risk factor for cadmium intoxication, suggesting that cadmium and iron share a common absorptive mechanism. Under investigation in this proposal is the premise that DMT1 is a complex H+-coupled and voltage-dependent ferrous-iron (Fe2+) transporter and that an acidic microclimate at the intestinal brush-border membrane provides the proton-motive force energizing DMT1. We will probe the molecular basis of H+-coupling and explore a physiological role for the significant uncoupled H+ fluxes (slippage) through DMT1 using the voltage clamp, radiotracer (55Fe) assays, and fluorescence approaches in Xenopus oocytes expressing wildtype DMT1 or mutant proteins. Next, we will examine iron transport in the mouse intestine using radiotracer and fluorescence approaches; the use of specific inhibitors of intestinal Na+/H+ exchangers (NHE3 and NHE2) and mutant mouse models lacking NHE3, NHE2, or the gastric H+/K+-ATPase will permit us to evaluate the roles of gastric acid and the intestinal brush-border acidic microclimate in providing the H+ to drive DMT1-mediated Fe2+ transport. The second premise is that DMT1 serves absorption not only of iron but also of certain other essential metals such as Co and Mn (as well as trace metals such as Ni and V) but that this promiscuity also makes it a major route of entry for the toxic heavy metal Cd. Here, we will determine the comprehensive substrate profile and metal-ion selectivity of DMT1 using our oocyte assays. Next, we will use the Belgrade anemic rat (which bears a mutation in DMT1) to examine the physiological significance of DMT1 in the absorption of each of the metals it is capable of transporting. Finally, we will identify metal-coordination sites in DMT1. Studying DMT1 in this way will lead to a better understanding of the mechanisms of nonheme iron absorption, the conditions required for efficient absorption, and the role of DMT1 in the metabolism of other transition metals. The results of this work will help drive development of new strategies for improving metal nutrition, or for treating iron overload and heavy-metal intoxication. Public Health Relevance: Iron deficiency leads to anemia and the hereditary disease hemochromatosis leads to toxic iron overload, illustrating the importance of balancing intestinal iron absorption. The gateway for dietary iron to enter the cells lining the intestine is a protein called DMT1, which can also transport the toxic metal cadmium. In this project we will study iron and cadmium transport via DMT1 at the molecular level to drive development of new strategies to improve iron nutrition and prevent the toxic effects of cadmium or iron overload.