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

汞对牙鲆噻嗪类敏感氯化钠协同转运蛋白的影响

• 批准号: 6660030
• 负责人: DAVID C DAWSON
• 金额: $ 17.41万
• 依托单位: MOUNT DESERT ISLAND BIOLOGICAL LAB
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6660030
• 关键词: Osteichthyes; Xenopus oocyte; animal genetic material tag; chloride channels; electrophysiology; intestines; ion transport; membrane permeability; membrane transport proteins; mercury; mercury poisoning; molecular cloning; protein sequence; radiotracer; saltwater environment; site directed mutagenesis; sodium chloride; thiazide; toxicant interaction; urinary bladder

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.

拟议研究的长期目标是了解 汞与膜转运相互作用的分子机制 proteins. 了解汞对活细胞的作用 由于存在许多潜在的目标和多个 汞的化学形式 定义特定毒性的机制 汞的影响将涉及确定（a）汞的活性形式， (b)细胞内的具体目标;（c）汞的接触途径 到这个目标。 这里提出的研究目的是利用 一个模型系统，比目鱼膀胱，研究相互作用 汞与特定的转运蛋白，噻嗪敏感， NaCl协同转运蛋白。 在以前的研究中，我们已经获得了证据， 与无机汞是一种特殊的， 可逆性噻嗪敏感协同转运蛋白阻断剂 膜的比目鱼膀胱，这种封锁导致 间接导致K分泌减弱。 拟议的实验 是基于汞的聚阴离子形式， 可能是HgCl 3 -1，是一种可逆的阻断剂， 协同转运蛋白，通过结合氯离子位点发挥作用， 协同转运蛋白 具体目标是：（1） 表征 汞与比目鱼尿中顶端NaCl协同转运蛋白的相互作用 膀胱 (2)为了描述汞对最近 克隆的比目鱼噻嗪敏感协同转运蛋白在非洲爪蟾中的表达 （3）利用定点突变技术， 参与结合的蛋白质结构域。 (4)探索其他 多阴离子形式汞的潜在目标，如CFTR和 Na/K/2Cl协同转运蛋白。 比目鱼的膀胱是一种独特的 这些研究的模型系统。 它是唯一的平板模型， 已知表达噻嗪敏感协同转运蛋白。 的机理 汞的作用也可能是独特的，因为它似乎涉及 一个非 共价可逆结合反应。 汞的相互作用 使用电生理学和放射性同位素表征 方法与协同转运蛋白的特定阻断剂结合。 放射性同位素的吸收和流出将用于表征 在非洲爪蟾卵母细胞中表达。 本研究结果 可以提供重要的见解，一个特定的分子机制， 汞对特定靶蛋白NaCl协同转运蛋白的作用 这对人体肾脏的远端盐重吸收很重要， 已知器官是汞中毒的部位。