Organization and Structure of ENaC Transmembrane Segments

ENaC 跨膜片段的组织和结构

• 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
• 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

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）：