Regulation of ENaC/degenerin channels by mechanical forces

描述(申请人提供)：施博士在获奖期间的职业目标是从她的导师那里获得科学上的独立性，并扩展她的实验技能：提高对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。在接受了两到三年的指导研究培训后，施正荣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员，开展可将研究成果转化为临床研究的研究，尤其是上皮性钠离子通道(ENaC)/变性蛋白家族离子通道介导的机械感觉。ENAC表达于全身许多上皮组织的顶膜。在对醛固酮敏感的远端肾单位，ENaC介导Na+吸收的限速步骤，因此对维持盐量平衡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，导致切应力和静水压力的变化，从而影响细胞的各种运输过程，包括对过滤后的Na+的吸收。ENAC活性随着切应力的增加而增加。ENaC/deenerin家族的其他成员也编码机械敏感离子通道，包括在秀丽线虫(C.elegans)中发现的通道。我们发现，与ENaCs类似，在异源表达系统中，特定的线虫通道(由MEC-4和MEC-10组成)被剪应力激活。我们之前的研究已经在ENaC亚基中发现了突变影响通道对剪切力反应能力的位置。基于这些发现和ENaC/deenerin家族相关成员的解析结构，我们假设在MEC-4/MEC-10通道的胞外区存在离散的构象变化，这些变化在通道开放过程中被传递到通道的孔洞中，以响应机械力。我们建议的研究将利用异源表达系统来确定MEC-4和MEC-10中的哪些位点/区域是该通道正确响应剪切力所必需的。选定的突变体将在线虫中表达，以探索这些突变体如何影响蠕虫的机械感知。成功完成这一应用中的拟议研究将促进我们对机械力如何调节ENaC/退行性离子通道的理解。

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.