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）：施博士在获奖期间的职业目标是培养独立于导师的科学独立性并扩大她的实验技能：提高对线虫进行行为测定的熟练程度以及电生理学和分子生物学技术方面的额外技术专长。经过 2 到 3 年的指导性研究培训后，石博士计划过渡到终身教职。她的长期职业目标是成为分子生物学和生理学广泛领域中完全独立的学术研究者，开展可将研究结果转化为临床研究的研究，特别关注上皮 Na+ 通道离子通道介导的机械感觉(ENaC)/退化蛋白家族。 ENaC 在全身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 Na+ 吸收的限速步骤，因此对于维持盐体积稳态和控制血压至关重要。肾小管上皮细胞受到不同的肾小管体积和流速的影响，导致剪切应力和静水压的变化，从而影响各种细胞运输过程，包括过滤的Na+的吸收。 ENaC 活性随着剪切应力的增加而增加。 ENaC/退化蛋白家族的其他成员也编码机械敏感离子通道，包括秀丽隐杆线虫 (C. elegans) 中发现的通道。我们发现，与 ENaC 类似，特定的线虫通道（由 MEC-4 和 MEC-10 组成）在异源表达系统中被剪切应力激活。我们之前的研究已经确定了 ENaC 亚基内的突变会影响通道响应剪切应力的能力的位点。基于这些发现以及 ENaC/degenerin 家族相关成员的解析结构，我们假设 MEC-4/MEC-10 通道的细胞外区域内存在离散的构象变化，这些变化在通道期间传递到通道的孔中响应机械力而打开。我们提出的研究将利用异源表达系统来识别 MEC-4 和 MEC-10 内通道正确响应剪切应力所需的位点/区域。选定的变体将在秀丽隐杆线虫中表达，以探索这些突变体如何影响蠕虫的机械传感。成功完成本申请中拟议的研究将增进我们对机械力如何调节 ENaC/简并蛋白离子通道的理解。