Regulation of ENaC/degenerin channels by mechanical forces

机械力对 ENaC/简并蛋白通道的调节

• 批准号: 8927629
• 负责人: Shujie Shi
• 金额: $ 13.82万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8927629
• 关键词: ASIC channel; Address; Affect; Aldosterone; Amiloride; Animal Model; Award; Behavior; Behavioral Assay; Biological; Biological Models; Blood Pressure; Caenorhabditis elegans; Cations; Cell membrane; Cells; Clinical Research; Complex; Distal; Duct (organ) structure; Epithelial; Epithelial Cells; Exhibits; Extracellular Fluid; Eyebrow structure; Faculty; Family; Gated Ion Channel; Goals; Hair; Health; Homeostasis; Hydrostatic Pressure; Indium; Ion Channel; Ions; Kidney; Knowledge; Mechanical Stimulation; Mechanics; Mediating; Mentors; Modeling; Molecular; Molecular Biology; Movement; Mutagenesis; Mutation; Nephrons; Neurons; Oocytes; Oryctolagus cuniculus; Peptide Hydrolases; Physiological; Physiology; Positioning Attribute; Probability; Proteins; Regulation; Renal tubule structure; Research; Research Personnel; Research Training; Rodent Model; Side; Signal Transduction; Site; Sodium; Sodium Channel; Sodium Chloride; Stimulus; Structure; System; Technical Expertise; Techniques; Testing; Tissues; Touch sensation; Translating; Transport Process; Tubular formation; Variant; Work; absorption; apical membrane; base; biological adaptation to stress; blood pressure regulation; career; epithelial Na+ channel; extracellular; in vivo; insight; member; mutant; patch clamp; polypeptide; receptor; response; shear stress; skills; uptake

DESCRIPTION (provided by applicant): Dr. Shi's career goals for the award period are to develop scientific independence from her mentor and broaden her experimental skills: developing proficiency in conducting behavioral assays in C. elegans and additional technical expertise in electrophysiological and molecular biological techniques. After 2 to 3 years of mentored research training, Dr. Shi plans to make the transition to a tenure-track faculty position. Her long-term career goals are to become a fully independent academic investigator in the broad fields of molecular biology and physiology, performing research that could translate research findings into clinical studies, with a particular focus on mechanosensation mediated by ion channels of the epithelial Na+ channel (ENaC) /degenerin family. ENaC is expressed at the apical membrane of many epithelial tissues throughout the body. In the aldosterone- sensitive distal nephron, ENaC mediates the rate-limiting step of Na+ absorption and thus is critical for maintaining salt-volume homeostasis and controlling blood pressure. Renal tubular epithelial cells are subjected to variable tubular volumes and flow rates, leading to changes in shear stress and hydrostatic pressure that affect a variety of cellular transport processes, including th absorption of filtered Na+. ENaC activity increases in response to increases in shear stress. Other members of the ENaC/degenerin family also encode mechanosensitive ion channels, including channels found in Caenorhabditis elegans (C. elegans). We found that, similar to ENaCs, specific C. elegans channels (comprised of MEC-4 and MEC-10) are activated by shear stress in a heterologous expression system. Our previous studies have identified sites within ENaC subunits where mutations affect the ability of the channel to respond to shear stress. Based on these findings and on the resolved structures of a related member of the ENaC/degenerin family, we hypothesize that there are discrete conformational changes within the extracellular region of MEC-4/MEC-10 channels that are transmitted into the channel's pore during channel opening in response mechanical forces. Our proposed studies will utilize a heterologous expression system to identify sites/regions within MEC-4 and MEC-10 that are required for the channel to respond properly to shear stress. Selected variants will be expressed in C. elegans in order to explore how these mutants affect mechanosensing in worms. Successful completion of proposed studies in this application will advance our understanding of how mechanical forces regulate ENaC/degenerin ion channels.

描述（由申请人提供）：Shi博士在奖励期间的职业目标是发展科学独立性，并扩大了她的实验技巧：在秀丽隐杆线虫进行行为测定方面的熟练程度以及电生理和分子生物学技术方面的其他技术专业知识。经过2至3年的指导研究培训，Shi博士计划过渡到终身教师职位。她的长期职业目标是成为分子生物学和生理学广泛领域的完全独立的学术研究者，进行研究，可以将研究结果转化为临床研究，特别关注由上皮Na+通道（ENAC） /degenerin家族的离子通道介导的机械感应。 ENAC在整个体内许多上皮组织的顶膜上表达。在醛固酮 - 敏感的远端肾单位中，ENAC介导了Na+吸收的速率限制步骤，因此对于维持盐量体内稳态和控制血压至关重要。肾小管上皮细胞受到可变的管状体积和流速，从而导致剪切应力和静水压力的变化，这些变化会影响各种细胞转运过程，包括TH吸收过滤的Na+。 ENAC活性随着剪切应力的增加而增加。 ENAC/Degenerin家族的其他成员还编码机械敏感的离子通道，包括在Caenorhabditis elegans（秀丽隐杆线虫）中发现的通道。我们发现，与ENAC相似，特定的秀丽隐杆线虫通道（由MEC-4和MEC-10组成）在异源表达系统中被剪切应力激活。我们以前的研究已经确定了ENAC亚基中突变影响频道对剪切应力反应能力的位点。基于这些发现以及ENAC/DeGenerin家族相关成员的分辨结构，我们假设MEC-4/MEC-10通道的细胞大细胞区域内存在离散的构象变化，这些变化在响应机械力的响应机械力时在通道开放期间传播到通道的孔中。我们提出的研究将利用一个异源表达系统来识别MEC-4和MEC-10中的位点/区域，这些位点/区域是该通道对剪切应力做出正确响应所需的。选定的变体将在秀丽隐杆线虫中表达，以探讨这些突变体如何影响蠕虫的机械连声。在本应用中成功完成拟议的研究将使我们对机械力如何调节ENAC/Degenerin离子通道的理解。