Transport Physiology of Molecular Biology of the Invertebrate Electrogenic 2 Na/1 H Antiporter

9317230 Ahearn这项研究是为了研究甲壳类动物胃肠道的离子传输过程，并将其与研究得更彻底的哺乳动物进行比较。在之前的研究中，我们已经在肠道和肾脏上皮细胞的刷状边界膜上发现了一种明显独特的无脊椎动物电致阳离子交换器，它参与钠和钙的平衡，并且在生理特性上与脊椎动物类似的转运蛋白不同。本项目将利用膜囊泡技术和分子生物学方法的结合来扩展这些早期研究，以表征这种独特的无脊椎蛋白在甲壳类动物的重要生物活动中的功能作用，如离子稳态和蜕皮。为了实现这些目标，将使用标准离心技术从龙虾（Homarus americanus）肝胰脏（配对后胃憩室）中制备纯化的刷状边缘和基底外侧质膜膜囊泡，以及与消化和钙调节活动相关的特定细胞内液泡衍生的膜。这些囊泡的运输性质将使用放射性同位素和分光光度技术的组合进行研究。龙虾电致阳离子交换剂的基因核苷酸序列将使用我们先前开发的单克隆抗体作为基因组探针来确定。从这些生理和分子研究中，我们应该能够阐明这种明显独特的无脊椎转运蛋白的功能作用，并将其基因序列与类似的，但生理上不同的哺乳动物Na+/H+反转运蛋白异构体进行比较。＊＊＊

9317230 Ahearn This investigation is a request for funds to study ion transport processes of the crustacean gastrointestinal tract and compare them to those of the more thoroughly investigated mammals. In previous studies, we have identified an apparently unique invertebrate electrogenic cation exchanger on the brush border membrane of gut and kidney epithelial cells which is involved in sodium and calcium balance and differs in physiological properties from analogous transport proteins from vertebrates. The present project will extend these early studies using the combination of membrane vesicle techniques and molecular biology procedures to characterize the functional role of this unique invertebrate protein in important biological activities of crustaceans such as ion homeostasis and moltin. To accomplish these objectives, purified membrane vesicles of brush border and basolateral plasma membranes and membranes derived from specific intracellular vacuoles that are associated with digestive and calcium regulatory activities will be prepared from lobster (Homarus americanus) hepatopancreas (paired diverticulum of the posterior stomach) using standard centrifugation techniques. Transport properties of these vesicles will be investigated using a combination of radioisotopic and spectrophotometric techniques. The gene nucleotide sequence of the lobster electrogenic cation exchanger will be determined using our previously developed monoclonal antibody as a probe of the genome. From these physiological and molecular studies we should be able to clarify the functional role(s) of this apparently unique invertebrate transporter and compare its gene sequence with those of analogous, but physiolgically distinct, mammalian Na+/H+ antiporter isoforms. ***