Organization and Structure of ENaC Transmembrane Segments

描述(由申请人提供)： 卡什兰博士目前正在匹兹堡大学克莱曼博士的实验室进行他的研究。他在获奖期间的职业目标是进一步发展他的批判性思维技能、实验室研究的新方法以及电生理、生化和分子生物学方法方面的技术专长。在克莱曼博士的实验室接受了一到两年的有指导的研究培训后，卡什兰博士计划过渡到终身教职的教职。他的长期职业目标是成为生物物理学和分子生理学广泛领域的完全独立的学术研究员，进行研究，深入了解影响临床问题的重要生物学问题，特别是蛋白质的结构和功能。这项研究的第一个具体目标的基础是我们实验室在上皮钠通道(ENaC)内的阿尔法亚单位(AlphaM1)的第一个跨膜片段上发现了一个相互作用表面。我们建议通过实验来鉴定与αM1相互作用的跨膜片段(S)，然后表征这两个片段之间的相互作用。为此，我们将开发一种新的报告试验来鉴定不同跨膜片段之间的相互作用，并进行互补突变和功能分析，以表征两个片段之间的相互作用。第二个具体目标是利用工程组氨酸和Ni(2)来表征ENaC内孔的结构，这可能有助于深入了解ENaC的门控、电导和离子选择性的机理。我们将在整个孔衬ENaC结构元素中进行扫描组氨酸突变，并使用切割的卵母细胞和从内向外切除的宏贴片技术来测量向突变通道的细胞内侧添加Ni(2)的效果。这项研究建议应该导致对ENaC孔的结构和功能的更好的理解，并可能对通道的选择性、电导和门控有更深入的了解。这里提出的这项研究可能会揭示Na()稳态的潜在机制，这种稳态的失败会导致血压变化和异常的粘液纤毛清除。

DESCRIPTION (provided by applicant): Dr. Kashlan is currently conducting his research in the laboratory of Dr. Kleyman at the University of Pittsburgh. His career goals for the award period are to further develop his critical thinking skills, new approaches towards laboratory research, and technical expertise in electrophysiological, biochemical, and molecular biological methods. After 1-2 years of mentored research training in Dr. Kleyman's laboratory, Dr. Kashlan plans to make the transition to a tenure-track faculty position. His long-term career goals are to become a fully independent academic investigator in the broad fields of biophysics and molecular physiology, performing research that gives insight into important biological problems that impact clinical issues, with a particular focus on the structure and function of proteins. The basis for the first specific aim of the proposed research is the discovery in our laboratory of an interaction surface on the first transmembrane segment of the alpha subunit (alphaM1) within the epithelial Na(+) channel (ENaC). We propose experiments to identify the transmembrane segment(s) that interact with alphaM1, and then to characterize the interaction between these two segments. To that end, we will develop a novel reporter assay to identify interactions between different transmembrane segments and perform complementary mutagenesis paired with functional assays to characterize the interaction between the two segments. The second specific aim will characterize the structure of the ENaC inner pore by utilizing engineered histidines and Ni(2+), which may give insights into the mechanisms of ENaC gating, conductance and ion selectivity. We will perform scanning histidine mutagenesis throughout pore lining ENaC structural elements and measure the effects of adding Ni(2+) to the intracellular side of the mutant channels using the cut open oocyte and excised inside-out macro patch techniques. This research proposal should result in a greater understanding of the structure and function of the ENaC pore, and may give insights into channel selectivity, conductance and gating. The research proposed here may inform mechanisms underlying Na(+) homeostasis, whose failure leads to alterations in blood pressure and abnormal mucociliary clearance.