Membrane Trafficking of Renal Potassium Channel

肾钾通道的膜运输

• 批准号: 6628486
• 负责人: Chou-Long Huang
• 金额: $ 30.26万
• 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 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628486
• 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（由申请人提供）：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 中的 K+ 分泌受膳食 K+ 摄入量和激素加压素的调节。研究表明，膳食 K+ 摄入量和加压素至少部分通过改变 CCD 中活性 K+ 通道的数量来改变 K+ 分泌。这种情况是如何发生的尚不完全清楚，但可能涉及通道膜运输的改变和/或沉默通道的激活。我们的长期目标是了解肾 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。我们的初步结果表明，ROMK 的内吞作用是通过网格蛋白包被的囊泡（CCV）介导的。该申请第一部分的具体目的是检查参与通道内吞作用的 ROMK 和 CCV 的结构元件，并检查低膳食 K+ 摄入量是否会通过增加内吞作用和随后的 ROMK 降解来减少 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。将使用爪蟾卵母细胞中的两电极电压钳和膜片钳记录以及 CCD 检查蛋白质相互作用的功能意义。我们还发现CCD顶膜中的一些ROMK通道被突触蛋白-1A抑制。该应用的第二部分是检验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。