EFFECT OF MERCURY ON THIAZIDE-SENSITIVE SODIUM CHLORIDE COTRANSPORTER IN FLOUNDER

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The long range objective of the proposed research is to understand the molecular mechanism by which mercury interacts with membrane transport proteins. Understanding the action of mercury on living cells is complicated by the existence of many potential targets and multiple chemical forms of mercury. Defining the mechanism of a specific toxic effect of Hg would involve identification of (a) the active form of Hg, (b) the specific target in the cell and (c) the route of access of Hg to this target. The object of the studies proposed here is to utilize a model system, the flounder urinary bladder, to study the interaction of mercury with a specific transport protein, the thiazide-sensitive, NaCl cotransporter. In previous studies we have obtained evidence consistent with the notion that inorganic mercury is a specific, reversible blocker of the thiazide sensitive cotransporter in the apical membrane of the flounder urinary bladder, and that this blockade leads indirectly to an attenuation of K secretion. The proposed experiments are based on the working hypothesis that a polyanionic form of Hg, perhaps HgCl3-1, is a reversible blocker of the thiazide-sensitive cotransporter that acts by binding to the chloride site on the cotransporter. Specific aims are: (1) To characterize the interaction of Hg with the apical NaCl cotransporter in flounder urinary bladder. (2) To characterize the effects of mercury on the recently cloned flounder thiazide-sensitive cotransporter expressed in Xenopus oocytes (3) To use the technique of site-directed mutagenesis to identify the domains of the protein involved in binding. (4) To explore other potential targets for polyanionic forms of Hg such as CFTR and the Na/K/2Cl cotransporter. The flounder urinary bladder serves as a unique model system for these studies. It is the only flat sheet model that is known to express the thiazide-sensitive cotransporter. The mechanism of action of Hg also may be unique in that it appears to involve a non- covalent, reversible binding reaction. The interaction of mercury will be characterized using both electrophysiological and radioisotope methodology in conjunction with specific blockers of the cotransporter. Radioisotope uptakes and effluxes will be used to characterize the transporter expressed in Xenopus oocytes. The results of this study could provide important insights into a specific molecular mechanism of action of mercury on a specific target protein, the NaCl cotransporter that is important in distal salt reabsorption in the human kidney, an organ known to be a site of mercury toxicity.