ATP-Sensitive K Channels in Renal Proximal Tubule

肾近端小管中 ATP 敏感 K 通道

• 批准号: 6895810
• 负责人: ALAN SEGAL
• 金额: $ 20.55万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6895810
• 关键词: Urodela; Xenopus oocyte; angiotensin II; arachidonate; basolateral membrane; biological signal transduction; body fluid osmolarity; electrophysiology; kidney function; laboratory mouse; molecular biology; phosphatidylinositols; polymerase chain reaction; potassium channel; receptor; renal tubular transport; renal tubule; sodium potassium exchanging ATPase; sulfonylurea; taurine; voltage /patch clamp

DESCRIPTION (provided by applicant): In the kidney, the majority of volume is reabsorbed in the proximal tubule (PT). The maintenance of unidirectional transepithelial Na transport in PT requires that K brought into the cell across the basolateral membrane (BLM) by the Na,K-ATPase pump be able to recycle back across the BLM through a K channel. The concept of "pump-leak coupling" refers to the increase in BLM K conductance (GK) that occurs in association with increased activity of the Na,K-ATPase pump. Indeed, a major question in renal physiology is understanding exactly how an increase in transepithelial transport leads to an increase in the dominant BLM K conductance. Despite its pivotal physiological role, relatively little is known about the in situ regulation of the dominant BLM K channel, and its molecular identity remains unknown. Here we plan to use a combination of electrophysiology and molecular biology to determine the molecular physiology and identity of this K channel in amphibian and mammalian PT. In Aim 1, single and ensemble K currents will be characterized using novel macropatch clamp recording from intact mouse PT testing the hypothesis that it is an ATP-sensitive K channel. Aim 2 explores the in situ behavior of the channel during modulation of transepithelial transport by major signaling pathways in the PT. We hypothesize that changes in transcellular Na flux, angiotensin II (Ang II), and arachidonic acid (AA) are critical regulators of transport, and therefore the K channel, in the PT. Aim 3 proposes to establish the molecular identity of this K channel using homology cloning combined with functional expression and antisense nucleotides in native salamander PT cells. The recombinant channel, which is hypothesized to be a heteromultimer comprised of a Kir6.1 subunit and a SUR2B sulfonylurea receptor, will be expressed and compared to the native channel in amphibian and mammalian PT. Success in these aims will deepen our understanding of salt and water handling in the PT and pave the way for subsequent structure-function studies with mutant channels. This work may also have important clinical implications, as therapeutic agents that interfere with the Ang II system (e.g., ACEIs, A2RBs) and AA (e.g., NSAIDS), which are among the most commonly prescribed medications. Therefore, new knowledge gained from the proposed studies are important in health, and will be highly relevant to the pathophysiology of hypertension, dysregulation of extracellular volume, and renal insufficiency.

描述（由申请人提供）：在肾脏中，大部分体积都在近端小管（PT）中重新吸收。 在PT中维持单向旋转的Na运输要求K通过Na，K-ATPase Pump将基底外侧膜（BLM）带入细胞，可以通过K通道在BLM上回收回收。 “泵裂耦合”的概念是指与Na，K-ATPase泵的活动增加有关的BLM K电导（GK）的增加。 实际上，肾脏生理学的一个主要问题是准确地了解旋转型运输的增加如何导致主要的BLM K电导率增加。 尽管它具有关键的生理作用，但对主要的BLM K通道的原位调节知之甚少，其分子身份仍然未知。 在这里，我们计划使用电生理学和分子生物学的组合来确定两栖动物和哺乳动物PT中该K通道的分子生理和身份。 在AIM 1中，将使用完整的小鼠PT录制的新型Macropatch夹具来表征单一和集合K电流，该记录测试了它是ATP敏感的K通道的假设。 AIM 2探讨了通过PT中的主要信号通路调制跨度传输时通道的原位行为。 我们假设跨细胞Na通量，血管紧张素II（ANG II）和花生四烯酸（AA）是PT中的关键调节剂，因此是K通道的关键调节剂。 AIM 3提出，使用同源性克隆结合了天然Salamander PT细胞中的功能表达和反义核苷酸，以建立该K通道的分子身份。 重组通道被认为是由Kir6.1亚基和SUR2B磺胺尿素受体组成的异杀剂，将表达并与两栖动物和哺乳动物PT中的天然通道进行比较。 这些目标的成功将加深我们对PT中的盐和水处理的理解，并为随后使用突变通道进行的结构功能研究铺平了道路。这项工作也可能具有重要的临床意义，因为干扰ANG II系统（例如ACEIS，A2RBS）和AA（例如NSAIDS）的治疗剂是最常见的药物之一。因此，从提出的研究中获得的新知识在健康中很重要，并且将与高血压的病理生理学，细胞外容量失调和肾功能不全相关。