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

描述（由申请人提供）：容量扩张或利尿治疗导致的远端流速升高增加了液体和电解质向远端肾单位的输送，导致刺激K+分泌，通常使血浆[K+]降至极低水平。已知“K+消耗”部分是由于高流速刺激远端肾单位中的K+分泌。然而，流动刺激K+分泌的细胞机制从未被理解。在这项提案中，我们计划解决这个问题，结合传统的体内和体外方法分析K+分泌和通道活性的新的分子敲除和基因转导策略。在哺乳动物肾脏中，K+在远端肾单位中分泌，远端肾单位包括连接小管和皮质集合管（CCD）的主细胞（PC），并且在基础条件下主要由内向整流K+通道（Kir1.1）介导。然而，最近的研究表明，大的钙激活的K+通道（BK）是流动刺激的K+分泌的途径。我们发现，BK-B1辅助亚基，已知提高钙敏感性和赋予cGMP-激酶激活的孔形成BK-a，是在兔和小鼠连接小管（CNT）的特异性表达。此外，我们发现BK-B1小鼠（与野生型对照组不同）未能增加K+排泄量，以响应体积扩张（VE）引起的流速增加。流量依赖性K+分泌已被证明在离体兔CNT暗示的内在机制，如拉伸或钙介导的激活BK。然而，当检查与体内微穿刺实验，远端流速与速率的K+分泌的关系有一个增加的斜率，这表明，其他外在介质，影响K+分泌从外面的CNT。一种可能的介质是一氧化氮，其在粗大的上行支中随着流速增加而释放，并且与犬中血流诱导的K排泄增加有关。因此，假设BK-B1亚基通过赋予BK-B1对Ca和/或NO/cGMP的敏感性来响应哺乳动物连接小管中的流速而增加K+分泌。该建议的假设基于该实验室最近的3个关键发现：1。BK-B1基因敲除小鼠未表现出流量依赖性K+分泌。2. BK-P1亚基在哺乳动物连接小管的顶端膜中特异性表达。3. BK-B1的存在增强了BK的钙敏感性，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：1.使用BK-B1基因敲除小鼠来确定BK-01亚基是否是流量依赖性n分泌所必需的。2.确定BK-B1亚单位在远端肾单位中的定位。3.通过基因转导确定小鼠连接小管中的BK-β 1是否是血流诱导的K+分泌所必需的。4.确定NO在血流诱导的小鼠远端肾单位K+分泌增加中的作用。敲除小鼠以确定BK-/31亚基是否是流量依赖性K+分泌所必需的。

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.