Mechanism of Polymodal Activation of VR1 Receptors

描述(申请人提供)：疼痛开始于周围神经末梢的特殊感觉神经元的转导，称为伤害性感受器。了解这些伤害性感受器如何对产生疼痛的刺激做出反应是了解疼痛生物学的关键一步。这项研究涉及伤害性感受的离子通道，特别是克隆的VR1受体，它可以检测和整合多种物理和化学刺激，包括热、香草素和酸。我们的目标是在分子水平上了解受体是如何作为一个多功能的有毒物质探测器对各种刺激做出反应的。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。我们的第一个目标是了解热是如何激活通道的。我们将确定热激活的能量格局，并探索受体中可能存在的热传感器及其结构基础。我们的第二个目标是研究辣椒素，辣椒素是如何激活这一通道的。我们将研究辣椒素结合、解绑和通道门控激活过程的生物物理特性，并确定它们的分子基础。我们将把热和辣椒素的激活途径联系起来，并检查物理和化学刺激检测是否使用了不同的机制。第三个目标是了解像酸这样的痛觉过敏介质是如何敏化通道的。我们将探索质子结合促进热和辣椒素激活的可能变构机制。第四个目标是研究通道的电压依赖性。我们将确定电压如何改变激活动力学，以及电压敏感度的来源。这项研究将提高我们对伤害性感觉转导的认识，并将有助于疼痛治疗的临床进展，特别是寻找具有全新作用模式和对伤害性感受器具有前所未有的选择性的止痛药。

DESCRIPTION (provided by applicant): Pain begins with transduction at peripheral nerve terminals of specialized sensory neurons called nociceptors. Understanding how these nociceptors respond to pain-producing stimuli is a key step towards the understanding of the biology of pain. This research concerns ion channels underlying nociception, in particular, the cloned VR 1 receptor, which detects and integrates multiple physical and chemical stimuli including heat, vanilloids and acids. Our goal is to understand, at the molecular level, how the receptor functions as a versatile noxious detector in response to various stimuli. Our approach involves patch-clamp recordings from recombinant channels in heterologous expression systems, combined with kinetic analysis to unravel the molecular events occurring during activation, along with mutagenesis to identify functional domains of the receptor. Our first objective is to understand how heat activates the channel. We will determine the energetic landscape of heat activation and explore the existence of possible heat sensors in the receptor and their structural basis. Our second objective is to investigate how capsaicin, the pungent ingredient in hot peppers, activates the channel. We will study the biophysical properties of the activation process regarding capsaicin binding, unbinding and channel gating, and determine their molecular basis. We will correlate heat and capsaicin activation pathways and examine whether different mechanisms are used for physical and chemical stimulus detection. The third objective is to understand how hyperalgesic mediators like acids sensitizes the channel. We will probe possible allosteric mechanisms by which proton binding promotes heat and capsaicin activation. The fourth objective is to investigate the voltage dependence of the channel. We will determine how the activation kinetics is altered by voltage and where the voltage sensitivity originates. The proposed research will improve our knowledge of nociceptive sensory transduction and will benefit clinical advances in pain therapy, in particular, the search for analgesic drugs that have an entirely new mode of action and an unprecedented selectivity for nociceptors.