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

这项为期4年的研究是为了继续研究甲壳类动物肝胰腺上皮细胞的离子转运机制。Ahearn博士之前的研究发现，在节肢动物和棘皮动物胃肠道上皮细胞的上皮刷状边界膜上，有一种明显独特的无脊椎动物阳离子交换剂，它对阿米洛胺敏感，产电，并将2 Na+或1 Ca2+交换为1 H+。该转运体参与了甲壳类动物肝胰腺的多种生物活动，包括：1)经上皮阳离子运输，2)质子分泌，3)蜕皮周期中细胞内钙储存，4)重金属解毒。这种运输系统是参与从食物中进入腔内钙的三种根尖蛋白之一，似乎也对潜在有毒重金属的吸收负责。电致刷状边界2na +/1H+交换器与电中性基底侧1Na+/1H+交换器一起监测细胞内pH和钠浓度。在本项目的后续研究中，他希望结合膜囊泡技术和分子生物学进一步表征2Na+/1H+和1Na+/1H+反转运蛋白在甲壳类动物肝胰腺生物学中的功能作用，并确定这两种无脊椎动物交换物的基因核苷酸序列。为了实现这些目标，他将在下一个资助周期内进行三个不同的研究方向：1)研究控制每种转运体功能的细胞调节机制，并与目前已知的哺乳动物Na+/H+交换器进行比较；2)应用PCR和表达克隆技术来确定这两个基因序列；3)继续研究该器官系统对二价阳离子的运输过程，通过描述重金属在刷状边界和基底外膜上的吸收机制，以及这些阳离子如何作为许多无脊椎动物共同解毒过程的一部分被隔离在线粒体和溶酶体中。通过这些生理和分子研究，他应该能够阐明这些反转运蛋白在离子转运调节和隔离中的功能作用，并将它们的基因序列与类似的哺乳动物Na+/H+反转运蛋白异构体的基因序列进行比较。

The proposed 4-year investigation is a request for funds to continue studying ion transport mechanisms of crustacean hepatopancreatic epithelial cells. Previous studies by Dr. Ahearn 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 1 Ca2+ 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 2 Na+/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, he would like to use a combination of membrane vesicle techniques and molecular biology to further characterize the functional 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. He 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 membranes 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, he 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.