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学、生物化学和分子生物学方法的技术专长。在 Kleyman 博士的实验室接受 1-2 年的指导研究培训后，Kashlan 博士计划过渡到终身教授职位。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者，开展研究以深入了解影响临床问题的重要生物学问题，特别关注蛋白质的结构和功能。本研究的第一个具体目标的基础是在我们的实验室中发现了上皮 Na(+) 通道 (ENaC) 内 α 亚基 (alphaM1) 第一个跨膜片段上的相互作用表面。我们提出实验来识别与 alphaM1 相互作用的跨膜片段，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基因测定法来识别不同跨膜片段之间的相互作用，并进行互补诱变与功能测定相结合，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 门控、电导和离子选择性的机制。我们将在整个孔衬里 ENaC 结构元件中进行扫描组氨酸诱变，并使用切开的卵母细胞和切除的由内而外的宏补丁技术测量将 Ni(2) 添加到突变体通道的细胞内侧的效果。这项研究计划应该会让人们更好地了解 ENaC 孔的结构和功能，并可能深入了解通道选择性、电导和门控。这里提出的研究可能会揭示Na(+)稳态的潜在机制，其失败会导致血压改变和粘液纤毛清除异常。

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.