ATP-Sensitive K Channels in Renal Proximal Tubule

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。在PT中维持单向跨上皮Na转运需要通过Na，K- atp酶泵通过基底外侧膜（BLM）进入细胞的K能够通过K通道在基底外侧膜上再循环。“泵-漏耦合”的概念是指与Na，K- atp酶泵活性增加相关的BLM K电导（GK）的增加。事实上，肾脏生理学的一个主要问题是准确理解上皮转运的增加如何导致显性BLM K电导的增加。尽管其具有关键的生理作用，但对显性BLM K通道的原位调节知之甚少，其分子特性仍不清楚。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特性。在Aim 1中，将使用新型巨膜片钳记录完整小鼠PT的单个和集合K电流，以验证它是atp敏感K通道的假设。目的2探讨了该通道在PT中通过主要信号通路调节跨上皮运输过程中的原位行为。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）的变化是运输的关键调节剂，因此K通道。Aim 3提出利用同源克隆结合功能表达和反义核苷酸在原生蝾螈PT细胞中建立这一K通道的分子特性。重组通道被假设为由Kir6.1亚基和SUR2B磺脲受体组成的异多聚体，将在两栖动物和哺乳动物的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，作为干扰Ang II系统的治疗药物（例如ACEIs， A2RBs）和AA（例如NSAIDS），这是最常用的处方药之一。因此，从所提出的研究中获得的新知识对健康非常重要，并且将与高血压、细胞外体积失调和肾功能不全的病理生理学高度相关。

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.