The overall objective of the proposed research is to identify and characterize the molecular targets and mechanisms of interaction of mercury with membrane transport proteins. We will focus specifically on the interactions of mercury with the Na-dependent taurine transport system in the hemoglobin containing coelomocytes (red cells, RBCs) of the marine polychaete, Glycera dibranchiata. This transport system is similar to taurine transport systems in mammalian tissues such as heart, kidney and brain. In Glycera RBCs, taurine is maintained at exceptional gradients (950:01; 190 mM intracellular taurine: 0.2 mM extracellular taurine). We have shown that this transport protein is very sensitive to inhibition by mercurial. A one minute exposure to 20 muM HgC12 inhibits taurine influx 50%. Studies using reduced sulfhydryl reagents of different molecular size indicated that the reactive sites on the transport protein appear to be partially occluded by the membrane since complete reversal is obtained only with small molecules. Since HgC12 in high chloride media is present predominantly as anionic complexes that are impermeant to membranes, we propose the operational hypothesis that the nonionic HgC12 complex is the form that reacts with taurine sulfhydryl groups lying in membrane spanning regions of the protein. We will test this hypothesis by addressing the following specific aims: (1) Physiologically characterize the form of HgC12 that interacts with the taurine transport system and identify the transport proteins involved in cell volume regulation that are sensitive to interaction with mercury. (2) Clone the Na-dependent taurine transporter from Glycera RBCs. (3) Characterize the interaction of mercurial with the taurine transporter expressed in Xenopus oocytes. Flux measurements on intact RBCs and on oocytes expressing the taurine transport protein will be done using radioisotope methods. The molecular procedures will depend on reverse transcription and PCR of poly(A)+RNA with taurine transport activity identified by expression of taurine transport activity of the mRNA fractions microinjected into Xenopus oocytes. Physiological studies will measure solute contents and net fluxes with a variety of techniques including ion selective electrodes, atomic absorption spectroscopy, HPLC and radioisotopic methods. The results of this study will provide important basic information on the molecular mechanism of interaction of mercury with the taurine transporter that has a hole in many animals tissues including human cardiac, nerve and kidney tissue.
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