Role of BK Channels in K+-driven Colonic Water Secretion in Health and Disease

项目摘要 本研究的目的是阐明结肠顶膜大电导钾通道（BK通道）在结肠癌发生、发展中的作用。 健康和疾病中的K+分泌。在感染性大肠杆菌中，肠水分泌是由增强的Cl- 分泌，而在常见的炎症性肠病，溃疡性结肠炎（UC），主要的运输缺陷 导致腹泻的是Na+吸收的显著减少或缺乏，这消除了腹泻的驱动力。 吸水率历史上，在活动性UC患者中报告了结肠K+分泌增加， 尽管其可能的临床意义和所涉及的机制尚未被研究。我们有 初步但明确的证据显示，结肠K+分泌增加， 顶端BK通道（介导K+从细胞中退出）和基底外侧Na-K-2Cl共- 在人UC和实验性UC中，NKCC 1转运蛋白（NKCC 1;其介导K+和水摄取到细胞中） 结肠炎模型。我们提出了UC的第二个新机制，即刺激结肠K+分泌可能 为UC患者结肠水分泌增加提供驱动力。因此，具体目标1是确定 BK通道介导的结肠K+分泌和水分泌在健康和疾病中的关系。我们 将通过使用在体结肠灌注、体外隐窝腺微灌注和Ussing室来实现这一点 实验，其将全部在大鼠中的正常和实验诱导的UC（DSS-结肠炎）中进行， 小鼠（IL-10敲除）结肠和正常和UC人结肠。具体目标2将解决分子 在正常和发炎的人类结肠（UC）内的活性K+分泌的机制。这将使我们能够 鉴定沿着表面隐窝细胞分布/表达的BKα剪接变体和BKβ（β1和β3）亚型 轴，并确定其同种型在正常和炎症（UC）大鼠和人结肠中的功能。其他研究 将专注于正常和发炎的人类结肠内的基底外侧K+和水摄取机制。具体 目的3将鉴定转录调节BK通道表达的促炎介质， UC结肠中BK通道的激活。在另外的研究中，BK通道对于合成和 促炎介质的分泌和使用BKα敲除的炎症发展，IL-10 敲除和BKα/IL-10双敲除小鼠模型。

PROJECT SUMMARY The aim of this project is to clarify the role of the apical membrane large conductance K+ (BK) channels in colonic K+ secretion in health and disease. In infective diarrheas, the intestinal water secretion is driven by enhanced Cl- secretion, whereas in the common inflammatory bowel disease, ulcerative colitis (UC), the main transport defect contributing to diarrhea is a marked decrease or absence of Na+ absorption, which removes the driving force for water absorption. Historically, an increased colonic K+ secretion has been reported in patients with active UC, although its possible clinical implications and the mechanisms involved have not been studied. We have developed preliminary, but clear-cut, evidence showing increases in colonic K+ secretion, in the expression of apical BK channels (which mediates K+ exit from the cells) and in expression of the basolateral Na-K-2Cl co- transporter (NKCC1; which mediates K+ and water uptake into the cells) in both human UC and an experimental model of colitis. We propose a second novel mechanism in UC, that stimulation of colonic K+ secretion may provide a driving force for increased colonic water secretion in UC. Thus, Specific Aim 1 is to determine the relationship between BK channel-mediated colonic K+ secretion and water secretion in health and disease. We will accomplish this by using in vivo colonic perfusion, in vitro crypt-gland micro-perfusion, and Ussing chamber experiments, which will all be performed in a normal and experimentally induced UC in rat (DSS-colitis) and mouse (IL-10 knockout) colon, and a normal and UC human colon. Specific Aim 2 will address the molecular mechanism of active K+ secretion within both a normal and an inflamed human colon (UC). This will allow us to identify the BKα splice variants and BKβ (β1 and β3) isoforms that distribute/express along the surface-crypt cell axis and establish which isoforms function in normal and inflamed (UC) rat and human colon. Additional studies will focus on basolateral K+ and water uptake mechanism(s) within a normal and inflamed human colon. Specific Aim 3 will identify the pro-inflammatory mediator(s) that regulate BK channel expression transcriptionally and activation of BK channels in UC colon. In additional studies, the importance of BK channel for the synthesis and secretion of proinflammatory mediators and for the development of inflammation using BKα knockout, IL-10 knockout and BKα/IL-10 double-knockout mouse models.