Investigating the stoichiometry and gating of DEG/ENaC channels with structural-functional studies

在感觉传导过程中，刺激被检测、处理并传递到神经系统。离子通道将物理刺激转化为电信号，是感觉传导的主要参与者。在触摸过程中，机械电转导（MeT）通道将机械刺激转化为电反应，并在秀丽隐杆线虫的触摸受体神经元（trn）中得到了很好的表征。在trn中形成MeT通道的蛋白质属于保守的DEG/ENaC/ASIC蛋白超家族，在神经元和其他组织中形成三聚体通道复合物。已知TRN MeT通道中存在两种DEG/ENaC蛋白；最近发现了第三个候选亚基，但其对体内功能的贡献尚不清楚。虽然它们的体内组成尚不清楚，但对于这些通道如何利用触摸携带的能量来催化通道激活或门控，人们所知的就更少了。为了研究MeT通道复合物的门控行为，我将使用以下实验策略：1。确定所有通道孔隙形成亚基：a)研究新的候选亚基缺失对触摸感觉的影响，并利用CRISPR/Cas9在孔隙区域进行工程突变。我将用FALCON装置描述这些突变对体内TRN机械转导的影响，FALCON装置将体内电生理与力反馈控制的机械刺激相结合。b)利用荧光标记亚基和荧光互补的成像技术研究MeT通道化学计量学，其中两个候选亚基被荧光团的一半标记。在这里，荧光的增加表明被标记的蛋白质非常接近。2. 确定某些通道域如何促进体内机械激活：我将使用FALCON装置来研究第二跨膜区域通道突变时MeT电流的变化，这对于通道功能至关重要。所获得的知识不仅将增强对秀丽隐杆线虫感觉机械转导的理解，还将增强对DEG/ENaC/ASIC通道家族其他成员的理解，以及我们对体内所有机械敏感离子通道如何响应触摸的基本理解。

During sensory transduction, stimuli are detected, processed and transmitted to the nervous system. Ion channels convert physical stimuli into electrical signals and are the leading actors in sensory transduction. During touch, mechanoelectrical transduction (MeT) channels transform mechanical stimuli into an electrical responses and are well characterized in the touch receptor neurons (TRNs) of the nematode Caenorhabditis elegans. The proteins forming the MeT channels in TRNs belong to the conserved superfamily of DEG/ENaC/ASIC proteins that form trimeric channel complexes in neurons and other tissues. Two DEG/ENaC proteins present in the TRN MeT channel are known; a third candidate subunit was recently discovered, but its contribution to in vivo function is unclear. While their in vivo composition is not known, even less is known about how these channels harness the energy carried by touch to catalyze channel activation or gating.To investigate the gating behavior of the MeT channel complex, I will use the following experimental strategy: 1. Identify all channel pore-forming subunits: a) Investigate the effect of the loss of the new candidate subunit on touch sensation and by engineering mutations in the pore region with CRISPR/Cas9. I will characterize the effect of these mutations on TRN mechanotransduction in vivo with the FALCON setup, which combines in vivo electrophysiology with force feedback-controlled mechanical stimulation. b) Investigate MeT channel stoichiometry with imaging techniques using fluorescence tagged subunits and fluorescence complementation, in which two candidate subunits are tagged with one half of a fluorophore. Here, an increase in fluorescence would indicate that the tagged proteins are in close proximity. 2. Determine how certain channel domains contribute to mechanical activation in vivo: I will use the FALCON setup to investigate the change in MeT currents upon channel mutations in the second transmembrane domain, which is known to be crucial for channel function.The gained knowledge will enhance the understanding not only of sensory mechanotransduction in C. elegans, but also of other members of the DEG/ENaC/ASIC channel family and beyond that our fundamental understanding of how all mechanosensitive ion channels respond to touch in vivo.