Regulation of ENaC/degenerin channels by mechanical forces

描述（由申请人提供）：施博士在奖励期内的职业目标是发展独立于导师的科学独立性，并拓宽她的实验技能：发展对秀丽隐杆线虫进行行为分析的熟练程度，以及在电生理和分子生物学技术方面的额外技术专长。经过2到3年的指导研究训练，施博士计划过渡到终身教职。她的长期职业目标是在分子生物学和生理学的广泛领域成为一名完全独立的学术研究者，进行研究，将研究成果转化为临床研究，特别关注上皮Na+通道(ENaC) /变性素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上表达。在醛固酮敏感的远端肾元中，ENaC介导Na+吸收的限速步骤，因此对维持盐容量稳态和控制血压至关重要。肾小管上皮细胞承受不同的小管体积和流速，导致剪切应力和静水压力的变化，影响各种细胞运输过程，包括过滤Na+的吸收。ENaC活性随着剪切应力的增加而增加。ENaC/degenerin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ENaCs类似，在异源表达系统中，特定的秀丽隐杆线虫通道（由MEC-4和MEC-10组成）被剪切应力激活。我们之前的研究已经确定了ENaC亚基中突变影响通道响应剪切应力能力的位点。基于这些发现和ENaC/degenerin家族相关成员的解析结构，我们假设MEC-4/MEC-10通道的细胞外区域存在离散的构象变化，这些构象变化在响应机械力的通道打开过程中传递到通道的孔中。我们提出的研究将利用异源表达系统来确定MEC-4和MEC-10中通道对剪切应力作出适当反应所需的位点/区域。选择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。成功完成本应用程序中提出的研究将促进我们对机械力如何调节ENaC/退化离子通道的理解。

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.