Molecular Basis of Sensory Transduction in C. elegans

描述(申请人提供)：拟议研究的长期目标是了解引起触觉和振动感觉的分子和细胞事件。这些和其他机械感觉对站立和行走以及控制膀胱功能和血压都是至关重要的。机械转导是这些感觉的第一步，但仍知之甚少。人们普遍认为，它依赖于离子通道(所谓的机电转换通道或MET通道)，离子通道因触摸携带的机械能而打开。在哺乳动物中形成MET通道的蛋白质仍然未知。最近，我们证明了MEC-4和MEC-10是线虫MET通道的致孔亚单位，负责线虫对低强度触摸的敏感性(O‘Hagan等，2005，Nat Neurosci 8：43)。既然线虫触觉感受器神经元中MET通道的分子特性已知，我们将寻求以下关键问题的答案：1)力如何从体表传递到MET通道；2)这种力如何导致通道开放。我们特别感兴趣的是了解触摸感受器神经元是如何检测力的，小到100纳米，被调整为主要对力的变化(振动)做出反应，并在不到1毫秒的时间内对刺激做出反应。提出了三个目标。首先，我们将确定膜变形是否足以激活重组线虫MET通道(目标1)，并探索如果存在的话，这种敏感性可能依赖于MEC-4和MEC-10的第二跨膜结构域中存在的保守序列基序(目标2)。下一步，我们将确定MET通道的体内激活是否涉及在压缩过程中可以作为能量存储设备的粘弹性元件(目标3)。最后，我们将结合体内电记录和微管束的超微结构分析，研究微管和微管束在力传递和放大中的作用(目标4A)，并研究微管和肌动蛋白结合蛋白在力传递中的作用。我们还建议开发和部署新的设备，用于控制机械刺激(力和位移)的应用，以及测量自由移动的线虫产生的力。这项拟议的研究结合了我们在感觉生物物理学、已识别线虫神经元的活体电子记录、遗传分析和超微结构研究方面的独特专业知识，以得出对触觉的深刻理解。从这些研究中学到的东西有可能提高人们对疾病和正常衰老中的触觉和功能障碍的理解。与公共健康相关：在正常衰老和糖尿病等慢性疾病的影响下，触觉和振动感受到损害。最近的估计表明，由于糖尿病和与年龄相关的触摸和振动感觉功能障碍而造成的医疗成本每年超过280亿美元。这项提议试图通过研究线虫来提高对触觉的理解，线虫是一种简单的动物，它的触觉比我们自己的更好地研究。从这项研究中学到的东西有可能为可能的触摸感觉退化的诊断工具和治疗方法提供新的见解。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand the molecular and cellular events that give rise to the senses of touch and vibration. These and other mechanical senses are critical for standing and walking as well as the control of bladder function and blood pressure. Mechanotransduction is the first step in each of these senses, but remains poorly understood. It is widely believed to rely on ion channels (so-called mechano-eletrical transduction or MeT channels) that open in response to the mechanical energy carried in a touch. The proteins that form MeT channels in mammals remain unknown. Recently, we demonstrated that MEC-4 and MEC-10 are pore-forming subunits of the MeT channel responsible for sensitivity to low-intensity touch in C. elegans (O'Hagan et al, 2005, Nat Neurosci 8: 43.). Now that the molecular identity of the MeT channel in C. elegans touch receptor neurons is known, we seek answers to the key questions of 1) how force is transferred from the body surface to MeT channels and 2) how such forces lead to channel opening. We are particularly interested in understanding how touch receptor neurons detect forces as small as 100nm, are tuned to respond primarily to changes in force (vibration) and respond to stimulation in less than 1 millisecond. Three aims are proposed. First, we will determine whether or not membrane deformation is sufficient to activate recombinant C. elegans MeT channels (Aim 1) and explore the possibility that such sensitivity, if present, might rely on a conserved sequence motif present in the 2nd transmembrane domain of MEC-4 and MEC-10 (Aim 2). Next, we will determine if in vivo activation of MeT channels involves visco-elastic elements that could act as energy storage devices during compression (Aim 3). Finally, we will investigate the role of microtubules and the microtubule bundle in force transfer and amplification (Aim 4A), pairing in vivo electrical recording with ultrastructural analysis of the microtubule bundle and investigate the contribution of microtubule- and actin- binding proteins to force transfer. We also propose to develop and deploy new devices for controlled application of mechanical stimuli (both force and displacement) and for measuring forces generated by freely moving C. elegans worms. The proposed research combines our unique expertise in sensory biophysics, in vivo electrical recording from identified C. elegans neurons, genetic analysis, and ultrastructural studies to derive a profound understanding of the sense of touch. What is learned from these studies has the potential to improve understanding of touch sensation and dysfunction in disease and normal aging. PUBLIC HEALTH RELEVANCE: The senses of touch and vibration are compromised in normal aging and by chronic diseases such as diabetes. Recent estimates suggest that the health costs due to diabetes- and age-related dysfunction of touch and vibration sensation are more than $28 billion annually. This proposal seeks to improve understanding of touch sensation by studying the roundworm C. elegans, a simple animal whose sense of touch is better studied than our own. What is learned from this research has the potential to provide new insight into possible diagnostic tools and treatments for the degradation of touch sensation.