Regulation of ENaC/degenerin channels by mechanical forces

描述（由申请人提供）：施博士在获奖期间的职业目标是发展科学独立于她的导师，并扩大她的实验技能：发展在C. elegans和电生理学和分子生物学技术方面的其他技术专长。经过2到3年的指导研究培训，施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的完全独立的学术研究者，进行可以将研究结果转化为临床研究的研究，特别关注上皮Na+通道（ENaC）/变性蛋白家族离子通道介导的机械感觉。ENaC在全身许多上皮组织的顶膜处表达。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可变的肾小管体积和流速的影响，导致剪切应力和静水压力的变化，影响各种细胞转运过程，包括过滤的Na+的吸收。ENaC活性响应于剪切应力的增加而增加。ENaC/简并蛋白家族的其他成员也编码机械敏感性离子通道，包括在秀丽隐杆线虫（Caelophabditis elegans）中发现的通道（C. elegans）。我们发现，类似于ENaCs，特异性C。elegans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亚基内的突变影响通道响应剪切应力的能力的位点。基于这些发现和ENaC/退化蛋白家族的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，这些构象变化在响应机械力的通道开放期间被传输到通道的孔中。我们提出的研究将利用异源表达系统来鉴定MEC-4和MEC-10内的位点/区域，这些位点/区域是通道对剪切应力适当响应所需的。选择的变体将以C表示。elegans，以探索这些突变体如何影响蠕虫的机械感应。成功完成本申请中的拟议研究将推进我们对机械力如何调节ENaC/简并蛋白离子通道的理解。

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.