Organization and Structure of ENaC Transmembrane Segments

ENaC 跨膜片段的组织和结构

基本信息

批准号：
7994908
负责人：
OSSAMA B KASHLAN
金额：
$ 5万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-16 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7994908
关键词：
Address Alveolus Amiloride Award Binding Sites Biochemical Biological Biological Assay Biophysics Blood Pressure Cations Clinical Data Descending colon Distal Diuretics Elements Engineering Epithelial Cells Epithelium Extracellular Domain Extracellular Fluid Faculty Failure Family Family member Goals Histidine Homeostasis Ions Laboratories Laboratory Research Liquid substance Lung Measures Mediating Membrane Mentors Methods Modeling Molecular Molecular Conformation Mucociliary Clearance Mutagenesis Na(+)-K(+)-Exchanging ATPase Nephrons Nociception Oocytes Oral cavity Physiology Play Positioning Attribute Potassium Potassium Channel Property Reporter Research Research Personnel Research Proposals Research Training Role Scanning Shapes Side Sodium Solvents Structure Surface Technical Expertise Techniques Thinking Tissues Universities apical membrane base blood pressure regulation career design epithelial Na+ channel extracellular insight member mutant novel novel strategies protein structure function research study skills urinary

项目摘要

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.

描述（由申请人提供）： Kashlan博士目前正在匹兹堡大学克利曼博士的实验室进行研究。他的奖项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，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进行了1 - 2年的指导研究培训之后，Kashlan博士计划过渡到终身教师职位。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，进行研究，以深入了解影响临床问题的重要生物学问题，并特别关注蛋白质的结构和功能。拟议研究的第一个具体目的的基础是，在我们实验室发现上皮Na（+）通道（ENAC）中Alpha亚基（Alpham1）的第一个跨膜段（Alpham1）的相互作用表面的发现。我们提出了实验，以识别与alpham1相互作用的跨膜段，然后表征这两个段之间的相互作用。为此，我们将开发一种新型的记者测定法，以识别不同跨膜段之间的相互作用，并执行与功能测定配对的互补诱变，以表征两个段之间的相互作用。第二个特定目的将通过利用工程组氨酸和Ni（2+）来表征ENAC内部孔的结构，这可能会深入了解ENAC门控，电导，离子和离子选择性的机制。我们将使用切开的开放式卵母细胞在突变通道的细胞内添加Ni（2+）的效果，并测量在整个孔内孔内部结构元件中进行扫描组氨酸诱变的作用，并切除内而外的宏观贴片技术。这项研究建议应该使对ENAC孔的结构和功能有更深入的了解，并可以深入了解渠道选择性，电导性和门控。此处提出的研究可能会为NA（+）稳态的基础机制提供信息，该机制的失败导致血压和异常粘膜清除率改变。