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

描述(申请人提供)：容量扩张或利尿治疗导致的远端血流速度增加，液体和电解质输送到远端肾单位，导致钾分泌刺激，通常使血浆[K]降至极低水平。已知“钾损耗”的部分原因是高流速刺激远端肾单位的钾分泌。然而，血流刺激钾分泌的细胞机制一直没有被了解。在这项提案中，我们计划使用新的分子敲除和基因转导策略结合传统的体内和体外方法来分析K分泌和通道活性，以解决这个问题。在哺乳动物的肾脏中，K由远端肾单位分泌，包括皮质集合管的连接小管和主细胞(PC)，在基础条件下主要通过内向整流钾通道(Kir1.1)来调节。然而，最近的研究表明，大的钙激活钾通道(BK)是流动刺激的钾分泌途径。我们发现，BK-B1辅助亚基在兔和小鼠的连接小管(CNT)中特异表达。BK-B1辅助亚基具有增强钙敏感性和激活成孔BK-a的cGMP-激酶的功能。此外，我们发现BK-B1小鼠(与野生型对照组不同)未能增加K的排泄，以响应体积扩张(VE)引起的流量增加。在分离的兔CNT中，已经证实了流量依赖性的K分泌，这表明了一种内在的机制，如拉伸或钙介导的BK的激活。然而，当用体内微穿孔实验检验时，远端流量与钾分泌速率的关系有一个增加的斜率，这表明其他外在介质正在影响来自CNT外部的钾分泌。1可能的介质是一氧化氮，它随着粗大的升肢流速的增加而释放，并与犬流行性钾排泄的增加有关。因此，推测BK-B1亚基通过增加钙和/或NO/cGMP对BK-B1的敏感性来增加K的分泌，以响应哺乳动物连接小管的流速。这一建议的假设是基于本实验室最近的三个关键发现：1.BK-B1基因敲除小鼠不表现出依赖于流量的K分泌。2.BK-P1亚基在哺乳动物连接小管的顶膜中特异表达。3.BK-B1的存在增强了BK的钙敏感性，是cGMP介导的BK激活所必需的。我们将通过以下具体目标来检验这一假说：1.使用BK-B1基因敲除小鼠来确定BK-01亚单位是否对流量依赖性n的分泌是必需的。2.确定BK-B1亚基在远端肾单位的定位。3.通过基因转导确定小鼠连接小管中的BK-β1是否是流导性钾分泌所必需的。4.确定NO在血流诱导的小鼠远端肾单位钾分泌增加中的作用。以确定BK-/31亚单位是否是依赖流动的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.