Temperature-Dependent Gating of Vanilloid Receptors

描述(申请人提供)：热感觉和疼痛使用离子通道来检测环境线索。热Trp通道是瞬时受体电位超家族的成员，是热刺激的主要探测器。与其他蛋白质相比，这些通道具有强烈的温度依赖性。这项研究的长期目标是了解通道是如何获得其强大的热敏感性的。我们将重点介绍香草素受体TRPV1，它是热Trp亚家族的创始成员。该通道在痛觉传导中起着关键作用，在外周感觉神经元中大量表达，似乎是检测和整合伤害性刺激的门户。我们之前的研究在确定TRPV1热敏感性的来源方面取得了进展，并表明该通道的N端含有模块化的热敏感域。我们建议利用这些发现对温度依赖门控的基本机制进行全面的生物物理研究，使用已被证明成功理解其他类型的离子通道门控的方法。目的1重点介绍热敏传感器的物理基础。需要检验的假设是，N-末端结构域与TRPV1同源物的不同温度表型有关。通过剖析表型差异的分子决定因素，我们将识别与温度敏感有关的残基和亚区。目的2研究感受域和亚单位之间的相互作用。我们将测试亚单位之间的热感应的协同性，描绘单个亚单位热感应事件对通道开放的贡献，并探索其他刺激对热敏感性的影响。这些结果将揭开TRPV1在广泛的温度范围内实现其生理功能的动态热敏感性的复杂机制。目标3解决热敏感域与沟道门的耦合问题。我们将测试整个通道的几个区域，并确定它们控制温度激活的变构机制。这一结果将阐明TRPV1中连接热敏和门控的温度门控路径。我们的方法包括从异源表达系统中的重组通道进行膜片钳记录，结合快速温度刺激和动力学分析来解开激活过程中发生的分子事件，并通过突变来确定受体的功能结构域。热Trp通道是开发新型止痛药的有吸引力的离子通道靶点，这种止痛药可以作用于产生疼痛的伤害性感受器。随着对这些通道如何发挥作用的深入了解，拟议的研究将有助于促进选择性药物的开发，用于治疗炎症、周围神经损伤、糖尿病和单纯疱疹引起的热痛觉过敏等病理疾病。

DESCRIPTION (provided by applicant): Thermal sensation and pain use ion channels for detection of environmental cues. Thermal TRP channels, members of the transient receptor potential superfamily, are the principal detectors of thermal stimuli. These channels have a steep temperature dependence compared to other proteins. The long term goal of this research is to understand how the channels obtain their strong thermal sensitivity. We will focus on the vanilloid receptor TRPV1, a founding member of the thermal TRP subfamily. The channel plays a pivotal role in pain transduction and is abundantly expressed in peripheral sensory neurons where it appears to act as a gateway for detection and integration of noxious stimuli. Our previous research made progress in identifying the origin of thermal sensitivity of TRPV1, and showed that the channel contains modular thermal sensor domains in its N-terminus. We propose to take advantage of these findings to perform a comprehensive biophysical study on the fundamental mechanisms of temperature-dependent gating, using approaches that have proven successful for understanding other types of ion channel gating. Aim 1 focuses on the physical basis of thermal sensors. The hypothesis to be tested is that the N-terminal domain is responsible for distinct temperature phenotypes of TRPV1 homologs. By dissecting the molecular determinants of the phenotypic differences, we will identify the residues and subdomains contributing to temperature sensing. Aim 2 examines the interactions between sensing domains and subunits. We will test the cooperativity of thermal sensing between subunits, delineate the contribution of individual subunit thermal sensing events to channel opening, and probe the influence of thermal sensitivity by other stimuli. The results will unravel complex mechanisms by which TRPV1 achieves a dynamic thermal sensitivity for its physiological function over broad temperature ranges. Aim 3 addresses the coupling of the thermal sensor domain with the channel gate. We will test several regions throughout the channel and determine the allosteric mechanisms by which they control temperature activation. The results will illuminate the temperature-gating pathway in TRPV1 that links thermal sensing and gating. Our approach involves patch-clamp recording from recombinant channels in heterologous expression systems, combined with fast temperature stimulation and kinetic analysis to unravel the molecular events occurring during activation, along with mutagenesis to identify functional domains of the receptor. Thermal TRP channels are attractive ion-channel targets for the development of novel analgesic drugs that could act peripherally at nociceptors where pain is generated. With insight into how the channels function, the proposed studies will help prompt the selective drug development for treatment of pathologies such as thermal hyperalgesia due to inflammation, peripheral nerve injury, diabetes and herpes simplex.