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

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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. ***