The overall objective is to identify molecular mechanisms and targets for mercury's impairment of plasma membrane transport systems in skate liver, specifically the proteins involved in cell volume regulation and taurine efflux. We recently demonstrated that hepatocytes isolated from the little skate (Raja erinacea) provide a powerful model system for studying the effects of mercury on mechanisms of cell volume regulation. Our studies indicate that changes in plasma membrane ion and solute permeability are sensitive and proximate events in the expression of mercury-induced cell injury, and provide direct support for the hypothesis that cell membrane is the target organelle. Furthermore, three decades that mercury exposure leads to cell swelling, our results demonstrated that mercury also prevents the normal regulatory volume decrease (RVD) observed after osmotic taurine transport, and increased Na+ permeability, two mechanisms that probably contribute to the impaired RVD. The focus of the proposed studies is to examine the molecular interactions of mercury with the volume-activated taurine transporter: 1. Define the driving forces, substrate specificity, and interaction of mercury with the taurine efflux transport proteins, using isolated basolateral plasma membrane vesicles. 2. Because C1-channels are thought to modulate volume-activated taurine transport and RVD in other cell types, we plan to examine the effects of HgC12 on C1- channel activity and taurine transport in skate hepatocytes using patch clamp techniques. 3. Test whether mercury is interfering with membrane recycling (exocytosis) in skate hepatocytes, and whether this is related to the inhibition of RVD, and 4. A long-term goal is the molecular cloning of the cDNA for the taurine efflux transport system in skate hepatocytes, and the identification of potential mercury binding sites (eg, cysteine residues) in the gene product. These studies should provide important insights into mechanisms of volume regulation and taurine homeostasis, and how mercury disrupts these fundamental cellular processes. Volume regulatory processes are required by all animal cells to counterbalance transmembrane oncotic gradients, but are particularly important for the survival of cells or organisms that must adapt to varying extracellular osmolarities.
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