Diarrheal Disease: A Physiologic Approach to Treatment

描述（由申请人提供）：该提议的基础是，迫切需要急性和慢性腹泻疾病的新药物治疗，这可能通过确定在基础状态和抑制状态下刺激刷状缘Na/H交换剂NHE 3的方法来实现，如大多数腹泻中所发生的那样。实现这种刺激的一个起点是了解NHE 3（至少在禁食状态下占肠道Na吸收的大部分）如何在正常情况下作为消化生理学的一部分进行调节，并在腹泻中受到抑制。在这个建议中，我们将扩大急性NHE 3调节的理解，通过检查我们发现既结合到NHE 3 C-末端，调节NHE 3活性的几种蛋白质的作用机制。这些蛋白质是1）CaM激酶II，2）CK 2，3）PLC 3。在该提议中要测试的主要假设是，NHE 3活性在基础和升高的Ca 2+条件下通过与这些C-末端结合配偶体的协调和动态相互作用来确定，这些C-末端结合配偶体不仅调节NHE 3，而且以协调的方式影响彼此与NHE 3的关联和调节。目的研究极化肠钠吸收细胞（Caco-2）中表达的这些蛋白质，并使用与生物化学方法（包括siRNA和药理学和分子生物学方法）相关的转运试验，（显性负性）抑制剂与分子修饰的NHE 3或缔合蛋白的结合，以确定NHE 3与这些支架蛋白的物理缔合对NHE 3基础的影响，刺激和抑制活性，并对参与其调节的NHE 3复合物。目的II测试的假设，即一个共同的结构域在NHE 3的C-末端与所有的蛋白质在目的I相关联，代表“开关”的一部分，它移动NHE 3之间来回的完全刺激状态，通过基础活性的抑制，这定义NHE 3的功能作为消化的一部分。这将通过确定目标I中研究的蛋白质与NHE 3的结合是否以可重复的和时间依赖性的方式随着NHE 3活性调节的变化而变化;这些NHE 3结合蛋白中的每一种的敲低和药理学/分子抑制剂是否改变NHE 3与其它相互作用蛋白的结合;以及包括通过诱变方法消除与这些缔合蛋白中的单个或多个的相互作用的结果。 公共卫生相关性：该提议的基础是，迫切需要治疗急性和慢性结肠炎疾病的新药，这可能通过确定在基础状态和抑制状态下刺激特定肠钠转运蛋白（刷状缘Na/H交换器NHE 3）的方法来实现，如在大多数结肠炎疾病中发生的。本研究探讨了几种蛋白质结合到NHE 3的细胞质部分影响其运输活性的基础条件下，当NHE 3被刺激或抑制模仿消化过程中发生的变化，并在消化道疾病的机制。此外，将检查这些蛋白质与NHE 3的协调关联，这些蛋白质都与NHE 3的小区域结合，以确定它们是否共同解释了NHE 3在消化和腹泻期间如何改变其活性。

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.