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.
