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

基本信息

批准号：
6301303
负责人：
DAVID C DAWSON
金额：
$ 3.56万
依托单位：
MOUNT DESERT ISLAND BIOLOGICAL LAB
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-01 至 2001-03-31
项目状态：
已结题

项目摘要

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重目标，情况变得复杂汞的化学形式。定义特定毒性的机制汞的影响将涉及鉴定 (a) 汞的活性形式， (b) 细胞内的特定目标和 (c) 汞的进入途径到这个目标。这里提出的研究目的是利用模型系统，比目鱼膀胱，用于研究相互作用汞与特定的转运蛋白，噻嗪类敏感的，氯化钠协同转运蛋白。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、顶端噻嗪类敏感协同转运蛋白的可逆阻断剂比目鱼膀胱的膜，这种封锁导致间接导致 K 分泌减弱。拟议的实验基于汞的聚阴离子形式的工作假设，也许 HgCl3-1 是噻嗪类敏感药物的可逆阻断剂通过与氯化物位点结合发挥作用的协同转运蛋白协同转运蛋白。具体目标是： (1) 表征牙鲆尿液中汞与顶端氯化钠协同转运蛋白的相互作用膀胱。 (2) 描述汞对近期环境的影响在非洲爪蟾中表达的克隆比目鱼噻嗪类敏感协同转运蛋白 (3)利用定点突变技术鉴定卵母细胞参与结合的蛋白质的结构域。 (4)探索其他汞的聚阴离子形式的潜在目标，例如 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。这是唯一的平板模型已知表达噻嗪类敏感的协同转运蛋白。其机制为汞的作用也可能是独特的，因为它似乎涉及非共价、可逆结合反应。汞的相互作用将使用电生理学和放射性同位素进行表征与协同转运蛋白的特定阻断剂结合的方法。放射性同位素的吸收和流出将用于表征转运蛋白在非洲爪蟾卵母细胞中表达。本研究结果可以为特定的分子机制提供重要的见解汞对特定目标蛋白（氯化钠协同转运蛋白）的作用这对于人类肾脏的远端盐重吸收很重要，已知是汞中毒部位的器官。