The Invertebrate 2Na+/1H+ Antiporter: Physiology, MolecularBiology, and Role in Heavy Metal Sequestration

无脊椎动物 2Na /1H 逆向转运蛋白：生理学、分子生物学以及在重金属封存中的作用

• 批准号: 9730874
• 负责人: Gregory Ahearn
• 金额: $ 16万
• 依托单位: University of Hawaii
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9730874&HistoricalAwards=false
• 关键词: Invertebrate; 2Na+; 1H+; Antiporter; Physiology

9730874 Ahearn The proposed investigation is a request for funds to continue to study ion transport mechanisms of crustacean hepatopancreatic epithelial cells. The previous studies identified an apparently unique invertebrate cation exchanger on the epithelial brush border membranes of gastrointestinal epithelial cells from arthropods and echinoderms which was amiloride-sensitive, electrogenic and exchanged either 2 Na+ or 1Ca2+ for 1 H+. This transporter was shown to be involved in a number of biological activities of the crustacean hepatopancreas that included 1) transepithelial cation transport, 2) proton secretion, 3) intracellular calcium storage during the molt cycle, and 4) heavy metal detoxification. This transport system was one of three apical proteins involved in the influx of luminal calcium from food and appears to be responsible for the uptake of potentially toxic heavy metals as well. The electrogenic, brush border 2Na+/1H+ exchanger works in conjunction with an electroneutral, basolateral 1Na+/1H+ exchanger to monitor intracellular pH and sodium concentration. In the proposed continuation of this project we would like to use a combination of membrane vesicle techniques and molecular biology to further characterize the function roles that the 2Na+/1H+ and 1Na+/1H+ antiporters have in crustacean hepatopancreatic biology and to determine the gene nucleotide sequences of these two invertebrate exchangers. Dr. Ahearn will undertake three different lines of research over the next grant cycle to attain these goals: 1) The investigation of cellular regulatory mechanisms controlling the functions of each transporter for comparison with what is currently known about Na+/H+ exchangers of mammals; 2) The application of PCR and expression cloning techniques to determine both gene sequences; and 3) The continued investigation of the transport processes for divalent cations by this organ system by describing the mechanisms for heavy metal uptake across the brush border and basolateral m embranes and how these cations are sequestered in mitochondria and lysosomes as part of a detoxification process common to many invertebrates. From these physiological and molecular studies, the PI should be able to clarify the functional role(s) of these antiporters in ion transport regulation and sequestration and compare their gene sequences with those of the analogous mammalian Na+/H+ antiporter isoforms.

9730874建议的调查是要求拨款，以继续研究甲壳类肝胰腺上皮细胞的离子转运机制。以往的研究发现，节肢动物和棘皮动物胃肠道上皮细胞上皮刷缘膜上存在一种明显独特的无脊椎动物阳离子交换器，该阳离子交换器对阿米洛利敏感，能产生电，可将2Na+或1Ca2+交换为1H+。该转运蛋白参与甲壳动物肝胰腺的多种生物学活动，包括1)跨上皮阳离子转运，2)质子分泌，3)蜕皮周期内钙储存，4)重金属解毒。这个转运系统是参与从食物中吸收钙的三种顶端蛋白之一，似乎也是吸收潜在有毒重金属的原因。电刷状边界2Na+/1H+交换器与电中和基侧1Na+/1H+交换器一起工作，以监测细胞内的pH和钠浓度。在本项目的继续工作中，我们将结合膜泡技术和分子生物学，进一步研究2Na+/1H+和1NA+/1H+逆向转运蛋白在甲壳类肝胰腺生物学中的功能作用，并确定这两个无脊椎动物转运蛋白的基因核苷酸序列。为了实现这些目标，埃亨博士将在下一个拨款周期内进行三个不同的研究方向：1)研究控制每个转运体功能的细胞调控机制，并将其与目前已知的哺乳动物的Na+/H+交换器的功能进行比较；2)应用聚合酶链式反应和表达克隆技术确定这两个基因的序列；3)继续研究二价阳离子在这个器官系统中的转运过程，方法是：1)描述跨越灌丛边界和基侧膜吸收重金属的机制，以及作为许多无脊椎动物共有的解毒过程的一部分，这些阳离子如何被隔离在线粒体和溶酶体中。通过这些生理和分子研究，PI应该能够阐明这些逆向转运蛋白在离子转运调节和隔离中的功能作用(S)，并将它们的基因序列与类似的哺乳动物Na+/H+逆向转运蛋白亚型进行比较。