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动物肝胰腺上皮细胞的离子转运机制而申请的。 Ahearn博士先前的研究在节肢动物和棘皮动物胃肠道上皮细胞的上皮刷状缘膜上发现了一种明显独特的无脊椎动物阳离子交换剂，该阳离子交换剂对阿米洛利敏感，产电，可将2 Na+或1 Ca 2+交换为1 H+。 该转运蛋白参与甲壳动物肝胰腺的许多生物学活性，包括1）跨上皮阳离子转运，2）质子分泌，3）蜕皮周期期间的细胞内钙储存，和4）重金属解毒。 该转运系统是三个顶端蛋白质之一，参与管腔钙从食物中流入，似乎是负责潜在的有毒重金属的吸收以及。 产电的刷状缘2 Na+/1H+交换器与电中性的基底外侧1 Na +/1H+交换器一起工作，以监测细胞内pH和钠浓度。 在该项目的拟议延续中，他希望使用膜囊泡技术和分子生物学的组合来进一步表征2Na+/1H+和1 Na +/1H+反向转运蛋白在甲壳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剂的基因核苷酸序列。 他将在下一个资助周期进行三个不同的研究方向以实现这些目标：1）控制每个转运蛋白功能的细胞调节机制的研究，以与目前已知的哺乳动物Na+/H+交换蛋白进行比较：2）应用PCR和表达克隆技术来确定两种基因序列;和3）的方法通过描述重金属穿过刷状缘和基底外侧膜的吸收机制，以及这些阳离子如何在线粒体和溶酶体中被隔离，作为许多人常见的解毒过程的一部分，继续研究该器官系统对二价阳离子的转运过程。无脊椎动物 从这些生理和分子研究中，他应该能够阐明这些反向转运蛋白在离子转运调节和螯合中的功能作用，并将它们的基因序列与类似的哺乳动物Na+/H+反向转运蛋白同种型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.