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: 6575668
• 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/6575668
• 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 蛋白质。了解汞对活细胞的作用是 由于存在许多潜在目标和多个 汞的化学形态。定义一种特定毒素的机制 汞的影响将包括识别(A)汞的活性形式， (B)牢房内的具体目标和(C)汞进入的路线 到这个目标。这里提出的研究的目的是利用 一个研究相互作用的模型系统--比目鱼膀胱 汞与一种特定的运输蛋白结合，对噻嗪敏感， 氯化钠共转运蛋白。在之前的研究中，我们已经获得了证据 与无机汞是一种特定的， 硫氮化物敏感共转运体的可逆性阻断剂 比目鱼膀胱膜，这种封锁导致 间接地导致钾分泌的减弱。拟议中的实验 都是基于汞的一种多阴离子形式的工作假设， 可能HgCl3-1是一种对噻嗪敏感的可逆性阻滞剂 协同转运蛋白，通过结合上的氯化位点起作用 辅助传送器。具体目标是：(1)描述 牙鲆尿汞与顶端氯化钠转运蛋白的相互作用 膀胱。(2)表征汞对新近环境的影响 在非洲爪哇中表达的甲硫肼敏感共转运蛋白的克隆 卵母细胞(3)利用定点诱变技术鉴定 参与结合的蛋白质结构域。(四)探索其他 多阴离子形式汞的潜在靶标，如CFTR和 Na/K/2Cl共转运蛋白。比目鱼的膀胱是一种独特的 为这些研究建立了模型体系。这是唯一的平板模型 已知表达对噻嗪敏感的共转运蛋白。它的作用机制 汞的作用也可能是独特的，因为它似乎涉及一种非 共价的可逆结合反应。汞的相互作用将 同时使用电生理和放射性同位素来表征 结合辅转运体的特定阻滞剂的方法学。 放射性同位素的吸收和流出将被用来表征 转运蛋白在非洲爪哇卵母细胞中的表达。这项研究的结果 可以提供对特定的分子机制的重要见解 汞对特定靶蛋白--氯化钠共转运蛋白的作用 这在人类肾脏的远端盐重吸收中是重要的， 已知的汞中毒部位的器官。