Structure and Function of TRP Thermosensation

描述(申请人提供)：研究目标是了解瞬时受体电位(Trp)通道是如何感知温度的。在这个项目中，中心假设是温度激活的Trp通道中独特的残基(结构域)使这些蛋白质能够感知温度的变化并通过质膜选通离子电流。利用大规模随机突变文库可以发现温度激活离子通道TRPM8和TRPV1的特定功能结构域。具体目标1：筛选热激活和冷激活离子通道TRPV1和TRPM8的大型突变库，以寻找专门影响温度激活而不是化学激活的突变。这一目标将测试在这些通道中是否存在温度激活特别需要的结构域。具体目标2：对这些突变体进行详细的电生理检查，以验证筛选结果，并找出温度突变是否解偶联了电压激活机制。这可能会揭示这个问题，如果电压激活是温度激活机制的基础，就像之前提出的那样，但仍然受到质疑。具体目标3：将已确定的残基突变为所有其他19个氨基酸，以了解哪些特定的分子相互作用与温度缺乏表型有关。这些实验可以给出详细的分子机制的见解，并导致关于温度激活机制的新的、可检验的理论。慢性疼痛与许多不同的疾病有关，而体温感觉是痛感的重要组成部分。因此，从分子水平上理解热感觉对疼痛领域是重要的和相关的。我预计这项研究将大大促进对我们如何在分子水平上感知温度的理解。

DESCRIPTION (provided by applicant): The research goal is to understand how transient receptor potential (TRP) channels sense temperature. In this project, the central hypothesis is that unique residues (domains) within the temperature-activated TRP channels enable these proteins to sense changes in temperature and to gate ion cun-ents through the plasma membrane. Specific functional domains of the temperature-activated ion channels TRPM8 and TRPV1 should be found by usage of large-scale random mutant libraries. Specific aim 1: screen large mutant libraries of the heat- and cold-activated ion-channels TRPV1 and TRPM8 to find mutations that specifically affect temperature-activation, but not chemical activation. This aim will test, if domains exist in these channels that are specifically required for temperature-activation. Specific aim 2: detailed electrophysiological examination of these mutants to verify screening results and to find out if temperature-mutations uncouple the mechanism of voltage-activation. This might shed light on the question, if voltage-activation underlies the mechanism of temperature-activation, as was previously proposed, but is still questioned. Specific aim 3: mutation of identified residues into all other 19 amino-acids to learn what specific molecular interactions are associated with the temperature-deficient phenotype. These experiments could give detailed insight into the molecular mechanism and lead to new, testable theories of the mechanism of temperature-activation. Chronic pain is associated with many different diseases and thermosensation represents a significant component of pain sensation. Therefore, a molecular understanding of thermosensation is important and relevant to the field of pain. I expect this study to significantly advance the understanding of how we sense temperature at the molecular level.