Role of BK-Beta 1 in flow-dependent K+ secretion in the CNT

描述（由申请人提供）：体积扩张或利尿剂治疗引起的远端血流速率升高，增加了液体和电解质向远端肾元的输送，导致K+分泌刺激，通常使血浆[K+]降至极低水平。众所周知，“K+消耗”部分是由于高流速刺激远端肾元分泌K+。然而，血流刺激的K+分泌的细胞机制从未被理解。在本提案中，我们计划利用新的分子敲低和基因转导策略，结合传统的体内和体外分析K+分泌和通道活性的方法来解决这个问题。在哺乳动物肾脏中，K+在远端肾元中分泌，其中包括皮质集管（CCD）的连接小管和主细胞（PC），并主要通过向内纠偏K+通道在基础条件下介导（Kir1.1）。然而，最近的研究表明，大的钙激活K+通道（BK）是血流刺激K+分泌的途径。我们发现BK-B1附属亚基，已知可提高Ca敏感性并赋予cgmp激酶活化形成孔的BK-a，在兔和小鼠连接管（CNT）中特异性表达。此外，我们发现BK-B1小鼠（与野生型对照不同）在体积扩张（VE）引起的流速增加时未能增加K+排泄。在分离的兔碳纳米管中已经证明了流动依赖的K+分泌，这表明其内在机制，如拉伸或钙介导的BK激活。然而，当用体内微穿刺实验检查时，远端流动速率与K+分泌速率的关系斜率增加，这表明其他外部介质正在影响碳纳米管外部的K+分泌。一种可能的介质是一氧化氮，它在粗大的上升肢中随着血流速率的增加而释放，并与犬血流诱导的钾排泄增加有关。因此，我们假设BK-B1亚基通过赋予Ca和/或NO/cGMP对BK-B1的敏感性来增加K+分泌，以响应哺乳动物连接小管的流速。该建议的假设基于该实验室最近的3个主要发现：1。BK-B1敲除小鼠没有表现出血流依赖性的K+分泌。2. BK-P1亚基在哺乳动物连接小管的顶膜中特异性表达。3. BK- b1的存在增强了BK对钙的敏感性，并且是通过cgmp介导的途径激活BK所必需的。我们将以以下具体目标来检验这一假设：使用BK-B1敲除小鼠来确定BK-01亚基对于血流依赖性n分泌是否必要。2. 确定BK-B1亚基在远端肾元的定位。3. 通过基因转导确定小鼠连接小管中的bk - β 1是否为血流诱导的K+分泌所必需。4. 确定NO在血流诱导的小鼠远端肾元K+分泌增加中的作用。敲除小鼠以确定BK-/31亚基是否为血流依赖性K+分泌所必需。

DESCRIPTION (provided by applicant): Elevated distal flow rates resulting from volume expansion or diuretic therapy increase the delivery of fluid and electrolytes to the distal nephron resulting in stimulation of K+ secretion, often reducing plasma [K+] to critically low levels. It is known that "K+ wasting" is due partially to high flow rates that stimulate K+ secretion in the distal nephron. However, the cellular mechanisms for flow-stimulated K+ secretion have never been understood. In this proposal, we plan to address this question using novel molecular knockdown and gene transduction strategies in combination with traditional in vivo and in vitro methods for analyzing K+ secretion and channel activity. In the mammalian kidney, K+ is secreted in the distal nephron, which includes the connecting tubules and principal cells (PC) of cortical collecting ducts (CCD) and is primarily mediated under basal conditions by inward rectifying K+ channels (Kir1.1). However, recent studies have shown that large Ca-activated K+ channels (BK) are the pathways for flow-stimulated K+ secretion. We found that the BK-B1 accessory subunit, known to heighten Ca sensitivity and confer cGMP-kinase activation of the pore-forming BK-a, was expressed specifically in the rabbit and mouse connecting tubules (CNT). Moreover, we found that BK-B1 mice (unlike the wild type controls) failed to increase K+ excretion in response to volume expansion (VE)-evoked increases in flow rate. Flow-dependent K+ secretion has been demonstrated in the isolated rabbit CNT suggesting an intrinsic mechanism, such as stretch or Ca-mediated activation of BK. However, when examined with in vivo micropuncture experiments, the relation for distal flow rate vs. rate of K+ secretion has an increased slope, suggesting that other, extrinsic mediators, are influencing K+ secretion from outside the CNT. 1 possible mediator is nitric oxide, which is released with increased flow rates in the thick ascending limb and has been implicated in the increase in flow-induced K excretion in dogs. Therefore, it is hypothesized that the BK-B1 subunit augments K+ secretion in response to flow rate in the mammalian connecting tubule by conferring Ca and/or NO/cGMP sensitivity to BK-B1. The hypothesis of this proposal is based on 3 recent key findings from this laboratory: 1. BK-B1 knockout mice do not demonstrate flow-dependent K+ secretion. 2. The BK-P1 subunit is expressed specifically in the apical membranes of mammalian connecting tubules. 3. The presence of the BK-B1 enhances the calcium sensitivity of BK and is necessary for activation of BK by cGMP-mediated pathways. We will examine this hypothesis with the following Specific Aims: 1. Use BK-B1 knockout mice to determine if the BK-01 subunit is necessary for flow-dependent n secretion. 2. Determine the localization of the BK-B1 subunit in the distal nephron. 3. Determine by gene transduction whether the BK-beta1 in the mouse connecting tubule is necessary for flow-induced K+ secretion. 4. Determine the role of NO in the flow-induced increase in K+ secretion in the mouse distal nephron. Knockout mice to determine if the BK-/31 subunit is necessary for flow-dependent K+ secretion.