Diarrheal Disease: A Physiologic Approach to Treatment

描述(申请人提供)：这项建议的基础是迫切需要新的药物治疗急性和慢性腹泻疾病，这可以通过确定在基础状态和抑制状态下刺激刷状边界Na/H交换NHE3的方法来实现，就像在大多数腹泻中发生的那样。实现这种刺激的一个起点是了解NHE3是如何调节的，NHE3至少在禁食状态下占肠道钠吸收的大部分，在正常情况下作为消化生理的一部分，在腹泻时是被抑制的。在这个提案中，我们将通过研究几种我们已经发现的既与NHE3 C末端结合又调节NHE3活性的蛋白质的作用机制来扩大对急性NHE3调控的理解。这些蛋白质是：1)CaM激酶II，2)CK2，3)PLC3。这一建议中要检验的主要假设是，NHE3的活性是在基础和升高的钙条件下通过与这些C末端结合伙伴的协调和动态相互作用来决定的，这些C末端结合伙伴不仅调节NHE3，而且以协调的方式影响彼此与NHE3的结合和调节。目的研究这些蛋白在极化的肠钠吸收细胞(Caco-2)中的表达，并利用siRNA、药物和分子(显性-负性)抑制剂与分子修饰的NHE3或结合蛋白等生化方法相关的转运分析来确定NHE3与这些支架蛋白的物理结合对NHE3的基础、刺激和抑制活性以及参与其调控的NHE3复合体的影响。AIM II验证了这样的假设，即NHE3 C末端的一个公共结构域代表着一个开关的一部分，该开关将NHE3在完全刺激状态和基础活性之间来回移动到抑制状态，这将NHE3定义为消化的一部分。这将通过确定目标I中研究的蛋白质与NHE3的关联是否随着NHE3活性调节的变化而以可重现和时间依赖的方式改变；这些NHE3关联蛋白中的每一种的击倒和药物/分子抑制剂是否改变NHE3与其他相互作用的蛋白质的关联；以及消除与单个或多个这些关联蛋白质的相互作用的后果，包括通过突变方法。 公共卫生相关性：这项建议的基础是迫切需要治疗急慢性腹泻疾病的新药，这可以通过确定在基础状态和抑制状态下刺激特定肠道钠转运蛋白-刷状边界Na/H交换NHE3的方法来实现，就像大多数腹泻疾病中发生的那样。这项研究考察了在基础条件下，以及当NHE3被刺激或抑制以模拟消化过程中和腹泻疾病中发生的变化时，与NHE3细胞质部分结合的几种蛋白质影响其转运活性的机制。此外，还将检查这些蛋白质与NHE3的协调联系，这些蛋白质都与NHE3的一小部分结合，以确定它们是否一起解释了NHE3在消化和腹泻过程中如何改变其活性。

DESCRIPTION (provided by applicant): The underpinning of this proposal is that there is an urgent need for new pharmacotherapy of acute and chronic diarrheal diseases, and this might be achieved by determining ways to stimulate the brush border Na/H exchanger NHE3 under both the basal state and in the inhibited state as occurs in most diarrhea. A starting point in achieving this stimulation is to understand how NHE3, which accounts for the majority of intestinal Na absorption at least in the fasting state, is regulated both normally as part of digestive physiology and as inhibited in diarrhea. In this proposal we will expand understanding of acute NHE3 regulation by examining the mechanisms of action of several proteins that we have found both bind to the NHE3 C-terminus and regulate NHE3 activity. These proteins are 1) CaM kinase II, 2) CK2, 3) PLC3. The major hypothesis to be tested in this proposal is that NHE3 activity is determined under both basal and elevated Ca2+ conditions by coordinated and dynamic interactions with these C-terminal binding partners, which not only regulate NHE3 but also affect each others' association with and regulation of NHE3 in a coordinated manner. Aim I studies each of these proteins expressed in polarized intestinal Na absorptive cells (Caco-2) and uses transport assays correlated with biochemical approaches including siRNA and pharmacologic and molecular (dominant-negative) inhibitors with molecularly modified NHE3 or the associating proteins to determine the consequences of NHE3 physical association with these scaffolded proteins on NHE3 basal, stimulated and inhibited activity and on NHE3 complexes involved in its regulation. Aim II tests the hypothesis that a common domain in the NHE3 C-terminus with which all of the proteins in Aim I associate, represents part of "a switch" which moves NHE3 back and forth between a fully stimulated state through basal activity to inhibition, which defines NHE3 function as part of digestion. This will be examined by determining whether association with NHE3 of the proteins studied in Aim I changes in a reproducible and time dependent manner with changes in regulation of NHE3 activity; whether knockdown and pharmacologic/molecular inhibitors of each of these NHE3 associating proteins alter NHE3 association with the other interacting proteins; and the consequences of eliminating interactions with single or multiple of these associating proteins including by mutagenesis approaches. Public Health Relevance: The basis of this proposal is that there is an urgent need for new drug treatment of acute and chronic diarrheal diseases, and this might be achieved by determining ways to stimulate a specific intestinal sodium transport protein, the brush border Na/H exchanger NHE3, under both the basal state and in the inhibited state as occurs in most diarrheal diseases. This study examines the mechanism by which several proteins which bind to the cytoplasmic part of NHE3 affect its transport activity under basal conditions and when NHE3 is stimulated or inhibited to mimic changes which occur during digestion and in diarrheal diseases. In addition, the coordinated association with NHE3 of these proteins which all bind to a small area of NHE3, will be examined to determine if together they explain how NHE3 changes its activity during digestion and in diarrhea.