Luminal proteolytic activation of collecting duct enac and atp-gated ppx receptors and its potential role in regulation of renal sodium reabsorption

上皮钠通道（ENaC）是一个跨膜离子通道，对钠（Na）具有高度渗透性。ENaC是远端肾元顶膜钠转运的关键调控因子；它在很大程度上决定了最终尿钠排泄，从而决定了身体钠平衡和全身血压。由于需要，它的功能受到各种局部和系统性因素的严格调节。最近，胰蛋白酶从收集管主细胞的管腔侧（顶细胞膜）起作用，已被证明可以提高体外ENaC活性；但其对肾钠重吸收的生理意义尚未见体内研究；尿液中含有多种可能在体内激活ENaC的蛋白酶。本研究申请的首要目的是在正常钠饮食（基线ENaC活性较低）、缺乏或存在胰蛋白酶抑制剂或ENaC阻阻剂阿米洛利的情况下，使用远端小管微注射技术（含放射性Na）直接评估腔内胰蛋白酶（作为一种原型蛋白酶）对ENaC功能的影响。有趣的是，一个被称为P2X受体的atp门控离子通道家族，也在收集管主细胞的顶端表达，具有与ENaC相似的结构，即它们有一个大的细胞外环，富含碱性氨基酸残基，包含Arg-X-X-Arg的一致序列，其中X是任何残基。目前还没有关于蛋白酶对P2受体活性影响的研究。因此，本学生申请的第二个目标是利用爪蟾卵细胞异种表达系统和双电极电压钳电生理技术，评估在ATP存在和不存在的情况下，细胞外蛋白酶对P2X受体活性的影响。我们将研究蛋白酶对收集管主细胞中发现的管腔P2X受体的影响（即4个同质和4个异质组合）。为了探索操纵ENaC蛋白酶激活的潜力（及其与P2X受体的相互作用-见下文），重要的是要有一系列可以模拟、抑制或增强蛋白酶活性的小分子化合物。该申请的第三个目的是在Discovery BioMed Inc （Birmingham, Alabama， USA）的实验室中进行，利用现代高通量生物测定方法在哺乳动物（主要是人类）细胞平台上筛选他们的化合物库（来自不同的合成和天然产品来源，并基于公司在肾脏生理学和生物化学方面的专业知识），以开发合适的药理药物。我们最近的研究表明，当腔内钠浓度高时，肾ENaC活性会被顶端P2X（4和/或4/6）受体的激活所抑制，当细胞外钠浓度降低时，这些受体会转变为ENaC活性的增强剂。我们已经提出这些P2X受体是Na传感器，负责ENaC活性的局部调节。有趣的是，在胰蛋白酶激活ENaC的原始研究中，阿米洛利并没有阻断胰蛋白酶对ENaC的作用，但在最近的一项研究中，它确实阻断了胰蛋白酶对ENaC的作用。这表明蛋白酶可能以多种方式改变ENaC功能，并提出了ENaC的另一种局部调节因子（如P2X）也可能是蛋白酶敏感的可能性。蛋白水解因子对P2X受体调控ENaC的影响尚未被研究。本应用程序的第四个目的是在正常或低钠饮食的大鼠中，利用大鼠分离开式收集管技术（采用全细胞膜片钳电生理），评估腔内蛋白酶对p2x介导的体外ENaC活性调节的影响，并测试上述确定的合适化合物。

The epithelial sodium channel (ENaC) is a transmembrane ion channel that is highly permeable to sodium (Na). ENaC is a key regulator of Na transport across the apical membrane of the distal nephron; it largely determines final urinary Na excretion, and thereby body Na balance and systemic blood pressure. By necessity, its function is tightly regulated through a variety of local and systemic factors. Recently, trypsin, acting from the luminal side (apical cell membrane) of collecting duct principal cells, has been shown to increase ENaC activity in vitro; however, there have been no in vivo studies to test its physiological significance for renal Na reabsorption; urine contains a variety of proteases that could potentially activate ENaC in vivo. The FIRST AIM of this studentship application is to assess directly the effect of luminal trypsin (as a prototypical protease) on ENaC function in vivo using the distal tubule micro-injection technique (with radioactive Na) in rats on a normal Na diet (when baseline ENaC activity is low), in the absence and presence of a trypsin inhibitor, or the ENaC blocker amiloride. Interestingly, a family of ATP-gated ion channels called P2X receptors, which are also expressed apically in the collecting duct principal cells, have a similar structure to ENaC, i.e. they have a large extracellular loop rich in basic amino acid residues that contain the consensus sequence of Arg-X-X-Arg, where X is any residue. As yet, there have been no studies on the effects of proteases on P2 receptor activity. Thus, the SECOND AIM of this studentship application is to assess the effect of extracellular proteases on P2X receptor activity in the presence and absence of ATP using the Xenopus oocyte heterologous expression system, and a twin-electrode voltage-clamp electrophysiological technique. We will examine the effects of proteases on those luminal P2X receptors found in the collecting duct principal cell (i.e. 4 homomeric and 4 heteromeric assemblies). To explore the potential to manipulate protease activation of ENaC (and its interaction with P2X receptors - see below), it is important to have a series of small molecule compounds that can mimic, inhibit, or potentiate, protease activity. The THIRD AIM of this application, to be undertaken in the laboratories of Discovery BioMed Inc (Birmingham, Alabama, USA), is to screen their compound libraries (which come from diverse synthetic and natural-product sources, and are based on the Company's expertise in renal physiology and biochemistry) using modern high-throughput bioassays on mammalian (primarily human)-cell-based platforms to develop suitable pharmacological agents. We have recently shown that renal ENaC activity is inhibited by activation of apical P2X(4 and/or 4/6) receptors when luminal concentrations of Na are high, and that these receptors switch to being potentiators of ENaC activity when concentrations of extracellular Na are lowered. We have proposed that these P2X receptors are Na sensors responsible for the local regulation of ENaC activity. Interestingly, in the original study of trypsin activation of ENaC, amiloride did not block trypsin's effect on ENaC, but in a more recent study it did. This suggests that proteases may act in more than one way to alter ENaC function, and raises the possibility that another local regulator (such as P2X) of ENaC might also be protease-sensitive. The effect of proteolytic factors on ENaC regulation by P2X receptors has not been investigated. The FOURTH AIM of this application is to assess the effect of luminal proteases on P2X-mediated regulation of ENaC activity in vitro using the rat isolated split-open collecting duct technique (with whole-cell patch clamp electrophysiology) in rats on a normal or low Na diet, and to test suitable compounds identified above.