Membrane Trafficking of Renal Potassium Channel

肾钾通道的膜运输

• 批准号: 7059374
• 负责人: Chou-Long Huang
• 金额: $ 25.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059374
• 关键词: Xenopus; Xenopus oocyte; apical membrane; cell membrane; clathrin; endocytosis; immunofluorescence technique; immunoprecipitation; intracellular transport; kidney; laboratory rat; potassium channel; protein protein interaction; protein purification; renal tubule; syntaxin; vasopressins

DESCRIPTION (provided by applicant): ROMK potassium (K+) channels are present in the apical membrane of principal cells of the kidney cortical collecting ducts (CCD) and are responsible for luminal K+ secretion in this nephron segment. K+ secretion in CCD is regulated by dietary K+ intake and the hormone vasopressin. It has been shown that dietary K+ intake and vasopressin alters K+ secretion, at least partly, by altering the number of active K+ channels in CCD. How this occurs is not fully understood, but may involve alteration of membrane trafficking of the channels and/or activation of silent channels. Our long-term objectives are to understand the molecular mechanism for membrane trafficking of renal K+ channels and its importance in physiological regulation of the channels. There are multiple pathways for endocytosis. Our preliminary results indicate that endocytosis of ROMK is mediated via clathrin-coated vesicles (CCVs). The specific aims of the first part of the application are to examine the structural elements of ROMK and CCVs involved in the endocytosis of the channels and to examine whether low dietary K+ intake decreases K+ channels by increasing endocytosis and subsequent degradation of ROMK. Association of ROMK with CCVs will be studied by immunofluorescent colocalization, biochemical purification, and immunocoprecipitation. Functional significance of protein interaction will be examined using two-electrode voltage clamp and patch-clamp recording in Xenopus oocytes as well as CCD. We also found that some of the ROMK channels in the apical membrane of CCD are inhibited by syntaxin-lA. The second part of the application is to test the hypothesis that vasopressin increases density of active channels by activating pre-existing silent channels. Biochemical binding assay and patch-clamp recording will be performed to examine this hypothesis.

描述（由申请方提供）：ROMK钾（K+）通道存在于肾皮质集合管（CCD）主细胞的顶膜中，负责该肾单位段的管腔K+分泌。CCD的K+分泌受膳食K+摄入量和激素加压素的调节。已经表明，膳食K+摄入和加压素至少部分地通过改变CCD中活性K+通道的数量来改变K+分泌。这是如何发生的还没有完全理解，但可能涉及通道的膜运输和/或沉默通道的激活的改变。我们的长期目标是了解肾脏K+通道膜运输的分子机制及其在通道生理调节中的重要性。内吞作用有多种途径。我们的初步研究结果表明，ROMK的内吞作用介导的网格蛋白包被囊泡（CCV）。本申请第一部分的具体目的是研究参与通道内吞作用的ROMK和CCV的结构元件，并研究低膳食K+摄入量是否通过增加ROMK的内吞作用和随后的降解来降低K+通道。将通过免疫荧光共定位、生化纯化和免疫共沉淀来研究ROMK与CCV的关联。蛋白质相互作用的功能意义将使用双电极电压钳和膜片钳记录在爪蟾卵母细胞以及CCD检查。我们还发现，在CCD的顶膜的一些ROMK通道被抑制突触融合蛋白-IA。本申请的第二部分是测试加压素通过激活预先存在的沉默通道来增加活性通道密度的假设。将进行生化结合测定和膜片钳记录来检验这一假设。