ATP-Sensitive K Channels in Renal Proximal Tubule

描述(申请人提供)：在肾脏，大部分体积被近端小管(PT)重新吸收。在PT中维持单向跨上皮钠转运需要通过Na，K-ATPase泵进入细胞基底膜(BLM)的K能够通过K通道通过BLM循环回来。“泵-漏耦合”的概念是指随着Na，K-ATPase泵活性的增加而发生的BLMK电导(GK)的增加。事实上，肾脏生理学中的一个主要问题是确切地理解跨上皮转运的增加如何导致主要的BLMK电导的增加。尽管BLMK通道具有重要的生理作用，但对它的原位调控知之甚少，其分子特性也尚不清楚。在这里，我们计划使用电生理学和分子生物学相结合的方法来确定两栖动物和哺乳动物PT中K通道的分子生理学和特性。在目标1中，将使用完整的小鼠PT的新型宏膜片钳记录来表征单个和整体K电流，以验证其是ATP敏感K通道的假设。目的2探讨在甲状旁腺主要信号通路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流量、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素，因此K通道也是如此。目的3利用同源克隆技术，结合功能表达和反义核苷酸，建立该钾通道的分子同源性。重组通道被认为是一个由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，并为随后的突变通道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，因为治疗药物干扰血管紧张素转换酶II系统(例如，ACEI、A2RBs)和AA(例如，非类固醇激素)，这是最常用的处方药物之一。因此，从拟议的研究中获得的新知识对健康非常重要，并将与高血压、细胞外容量调节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.