Sodium Entry into Amiloride-Sensitive Epithelia

描述(申请人提供)：钠重吸收上皮细胞，如肾、远端和集合管，具有控制全身钠平衡的主要功能。这些上皮细胞含有被利尿剂阿米洛利抑制的顶膜Na+通道。正是在这些通道的水平上，发生了维持Na+稳态所需的反馈控制机制。该项目的长期目标仍然是在分子水平上阐明通过这些传导进入途径调节离子流动的机制。在前一批款期内，提出了四项新的意见，构成了这项延续申请的基础。首先，我们发现钙离子参与了肌动蛋白与ENaC相互作用后对电导的影响。第二，在α-ENaC的C-末端有一个很短的14-AA片段，这对肌动蛋白与ENaC的功能相互作用是至关重要的。第三，我们已经确定了ENaC、Synaxin和其他新的细胞质调节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量的纯净、功能完整的α-ENaC。因此，我们建议1)检验t-SNARs(例如，Synaxin 1A)和Annexins直接调节ENaC功能的假设；2)检验肌动蛋白直接与ENaC结合的假设，从而诱导构象变化导致通道电导和阳离子选择性的变化。我们将确定ENaC与Synaxin、肌动蛋白、膜联蛋白和其他细胞骨架连接元件(如Ezrin)之间的物理接触位置。调节合成素活性的蛋白质，如SNAP 23/25和MUNC-18，也将被检测它们对合成素-ENaC相互作用的功能和物理影响。我们还将结晶a-ENaC，目的是提供这个亚基的详细分子图像。这些结果将提供对阿米洛利敏感的钠离子通道的性质和调控的新见解，以及这些通道与细胞骨架相互作用和被细胞骨架调制的方式，并提供这些重要离子通道的第一个近原子水平的细节。因此，了解ENaC调节、传导和选择性的分子基础将为治疗日益增多的ENaC相关疾病提供独特的机会。

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.