Sodium Entry into Amiloride-Sensitive Epithelia

钠进入阿米洛利敏感上皮细胞

• 批准号: 6776481
• 负责人: DALE J BENOS
• 金额: $ 33.67万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6776481
• 关键词: Xenopus; Xenopus oocyte; actins; amiloride; annexins; binding sites; biological transport; conformation; crystallization; diuretics; epithelium; protein protein interaction; protein structure function; renal tubular transport; saluresis; site directed mutagenesis; sodium channel; surface plasmon resonance; syntaxin

DESCRIPTION (provided by applicant): Sodium reabsorbing epithelia, such as renal, distal, and collecting tubules, have as their major function the control of whole-body sodium balance. These epithelia contain apical membrane Na + channels that are inhibited by the diuretic amiloride. It is at the level of these channels that the feedback control mechanisms necessary for the maintenance of Na + homeostasis occur. The long-term goal of this project remains to elucidate at the molecular level the mechanisms responsible for the regulation of ion flow through these conductive entry pathways. During the previous grant period four novel observations were made that form the basis of this continuation application. First, we discovered that Ca 2v was involved in the effect on conductance following the interaction of actin with ENaC. Second, a short, 14-aa segment in the C-terminal of alpha-ENaC was identified as being crucial for actin's functional interaction with ENaC. Third, we have identified new functional and physical interactions between ENaC, syntaxin, and other novel cytoplasmic regulatory elements. Fourth, we have utilized the baculovirus system to produce milligram quantities of pure, functionally intact alpha-ENaC. Therefore, we propose to 1) test the hypothesis that t-SNARES (e.g., syntaxin 1A) and annexins directly modulate ENaC function; and 2) test the hypothesis that actin directly binds to ENaC, thereby inducing a conformational change resulting in changes in channel conductance and cation selectivity. We will identify the site of physical contact between ENaC and syntaxin, actin, annexins, and other cytoskeletal linking elements such as ezrin. Proteins that regulate the activity of syntaxin, such as SNAP 23/25 and munc-18, will also be examined for their functional and physical influences on syntaxin-ENaC interactions. We will also crystallize a-ENaC with the goal of providing a detailed molecular picture of this subunit. These results will offer new insights into the nature and regulation of amiloride-sensitive Na+ channels, the ways that these channels interact with and are modulated by the cytoskeleton, and provide the first near atomic-level detail of these important ion channels. Thus, understanding the molecular basis for ENaC regulation, conduction, and selectivity will provide unique opportunities for therapeutic interventions in an ever-increasing plethora of ENaC-related diseases.

描述（由申请人提供）：钠重吸收上皮，如肾小管、远端小管和集合小管，其主要功能是控制全身钠平衡。这些上皮细胞含有被利尿剂阿米洛利抑制的顶端膜Na +通道。正是在这些通道的水平上，发生了维持Na +稳态所必需的反馈控制机制。该项目的长期目标仍然是在分子水平上阐明离子通过这些导电进入途径的调节机制。在上一个赠款期间，提出了四项新的意见，构成了这次继续申请的基础。首先，我们发现ca2v参与了肌动蛋白与ENaC相互作用后对电导的影响。其次，在α -ENaC的c端发现了一个短的14-aa片段，对于肌动蛋白与ENaC的功能相互作用至关重要。第三，我们已经确定了ENaC、syntaxin和其他新的细胞质调控元件之间新的功能和物理相互作用。第四，我们利用杆状病毒系统生产了毫克级的纯，功能完整的α - enac。因此，我们建议：1)验证t-SNARES（例如syntaxin 1A）和annexin直接调节ENaC功能的假设；2)验证肌动蛋白直接与ENaC结合，从而引起构象变化，从而改变通道电导和阳离子选择性的假设。我们将确定ENaC与syntaxin， actin， annexin和其他细胞骨架连接元件（如ezrin）之间的物理接触位点。调节syntaxin活性的蛋白质，如SNAP 23/25和munc-18，也将被研究它们对syntaxin- enac相互作用的功能和物理影响。我们还将结晶a- enac，目的是提供该亚基的详细分子图。这些结果将为了解酰胺敏感Na+通道的性质和调控、这些通道与细胞骨架相互作用和调节的方式提供新的见解，并提供这些重要离子通道的第一个接近原子水平的细节。因此，了解ENaC调控、传导和选择性的分子基础将为日益增多的ENaC相关疾病的治疗干预提供独特的机会。