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，大部分体积在近端小管（PT）中被重吸收。 PT 中单向跨上皮 Na 转运的维持需要由 Na,K-ATP 酶泵穿过基底外侧膜 (BLM) 进入细胞的 K 能够通过 K 通道穿过 BLM 循环回来。 “泵漏耦合”的概念是指 BLM K 电导 (GK) 的增加与 Na,K-ATP 酶泵活性的增加相关。 事实上，肾脏生理学的一个主要问题是准确理解跨上皮转运的增加如何导致主要 BLM K 电导的增加。 尽管其具有关键的生理作用，但人们对占主导地位的 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，我们计划结合电生理学和分子生物学来确定两栖动物和哺乳动物 PT 中该 K 通道的分子生理学和身份。 在目标 1 中，将使用来自完整小鼠 PT 的新型大膜片钳记录来表征单个和整体 K 电流，测试其是 ATP 敏感 K 通道的假设。 目标 2 探索 PT 中主要信号传导途径调节跨上皮转运过程中通道的原位行为。 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) 的变化是 PT 中转运的关键调节因子，因此也是 K 通道的重要调节因子。 目标 3 建议使用同源克隆结合天然蝾螈 PT 细胞中的功能表达和反义核苷酸来确定该 K 通道的分子身份。 重组通道被假设为由 Kir6.1 亚基和 SUR2B 磺酰脲受体组成的异多聚体，将在两栖动物和哺乳动物 PT 中表达并与天然通道进行比较。 这些目标的成功将加深我们对 PT 中盐和水处理的理解，并为后续突变通道的结构功能研究铺平道路。这项工作也可能具有重要的临床意义，因为治疗剂会干扰 Ang II 系统（例如 ACEI、A2RB）和 AA（例如 NSAIDS），而这些都是最常用的处方药物。因此，从拟议的研究中获得的新知识对健康很重要，并且与高血压的病理生理学、细胞外容量失调和肾功能不全高度相关。