Organization and Structure of ENaC Transmembrane Segments

描述（由申请人提供）： 博士Kashlan目前正在匹兹堡大学Kleyman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能力，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者，进行研究，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。所提出的研究的第一个具体目标的基础是在我们的实验室中发现上皮Na（+）通道（ENaC）内α亚基（alphaM 1）的第一个跨膜段上的相互作用表面。我们提出的实验，以确定与alphaM 1相互作用的跨膜段（S），然后表征这两个部分之间的相互作用。为此，我们将开发一种新的报告基因检测，以确定不同的跨膜片段之间的相互作用，并进行互补诱变与功能检测配对，以表征两个片段之间的相互作用。第二个具体目标是通过利用工程化组氨酸和Ni（2+）来表征ENaC内孔的结构，这可能会对ENaC门控、电导和离子选择性的机制提供见解。我们将在整个孔内衬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+）添加到突变通道的细胞内侧的影响。这项研究建议应该会导致对ENaC孔的结构和功能有更深入的了解，并可能深入了解通道选择性，电导和门控。本文提出的研究可能为Na（+）稳态的潜在机制提供信息，Na（+）稳态的失败导致血压和粘膜纤毛清除异常的改变。

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.