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

BK-Beta 1 在 CNT 流依赖性 K 分泌中的作用

• 批准号: 7212856
• 负责人: STEVEN SANSOM
• 金额: $ 27.12万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7212856
• 关键词: Address; Adenoviruses; Arginine; Attenuated; Bartter Disease; Brain; Calcium; Calcium-Activated Potassium Channel; Canis familiaris; Cell model; Cells; Condition; Coupling; Cultured Cells; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Distal; Distal convoluted renal tubule structure; Diuretics; Duct (organ) structure; Electrolytes; Epithelial Cells; Excretory function; Gene Transfer; Genes; ITPR1 gene; In Vitro; Intercalated Cell; KCNJ1 gene; Kidney; Knockout Mice; Laboratory Finding; Limb structure; Liquid substance; Localized; MDCK cell; Mediating; Mediator of activation protein; Methods; Micropuncture; Modeling; Molecular; Mus; NG-Nitroarginine Methyl Ester; Nephrons; Nitric Oxide; Oryctolagus cuniculus; Pathway interactions; Plasma; Play; Potassium; Potassium Channel; Property; Protein Isoforms; Rate; Recycling; Renal tubule structure; Role; Scheme; Signal Transduction; Smooth Muscle; Stretching; Thiazide Diuretics; Thick; Tissues; Transduction Gene; Transfection; Tubular formation; apical membrane; base; in vivo; inhibitor/antagonist; large-conductance calcium-activated potassium channels; novel; promoter; research study; response; shear stress; wasting

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.

描述（由申请人提供）：由于体积膨胀或利尿治疗而导致的远端流量升高增加了流体和电解质向远端肾单位的输送，从而导致K+分泌的刺激，通常会将血浆[K+]降低到严重的低水平。众所周知，“ k+浪费”部分是由于刺激远端肾单位的K+分泌的高流速。但是，从未理解过流动刺激的K+分泌的细胞机制。在此提案中，我们计划使用新型的分子敲低和基因转导策略与传统的体内和体外方法结合使用，以分析K+分泌和通道活性。在哺乳动物的肾脏中，K+在远端肾单位中分泌，其中包括皮质收集管（CCD）的连接小管和主要细胞（PC），主要是通过向内介导的基础条件介导的K+通道（KIR1.1）。但是，最近的研究表明，大型CA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，已知的Ca敏感性并赋予孔形成BK-A的CGMP-激酶激活，在兔子和小鼠连接小管（CNT）中专门表达。此外，我们发现BK-B1小鼠（与野生型控制不同）未能增加K+排泄，以响应体积膨胀（VE）引起的流速增加。在分离的兔CNT中已经证明了流动依赖性的K+分泌，该兔CNT提出了内在机制，例如拉伸或CA介导的BK激活。但是，当通过体内微源实验进行检查时，远端流速与K+分泌速率的关系具有增加的斜率，这表明其他外部介质介质会影响CNT以外的K+分泌。 1可能的介体是一氧化氮，在较厚的升肢中流速增加，该氧化物被释放，并且与狗流动诱导的K排泄的增加有关。因此，假设BK-B1亚基通过赋予Ca和/或/或/CGMP对BK-B1的敏感性来响应哺乳动物连接小管的流量增强K+分泌。该提案的假设基于该实验室的最新主要发现：1。BK-B1敲除小鼠没有证明依赖流动的K+分泌。 2。BK-P1亚基在哺乳动物连接小管的顶端膜中特别表达。 3。BK-B1的存在增强了BK的钙敏感性，对于通过CGMP介导的途径激活BK是必需的。我们将以以下特定目的检查这一假设：1。使用BK-B1敲除小鼠来确定BK-01亚基是否对于流量依赖性n分泌是必需的。 2。确定BK-B1亚基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-BETA1是否对于流动诱导的K+分泌是必需的。 4。确定NO在小鼠远端肾单位中的K+分泌增加中的作用。敲除小鼠以确定BK-/31亚基是否需要流动依赖性K+分泌。