Mechanisms of TRPV1 channel gating and modulation by temperature and vanilloids

我的长期目标是建立一个独立的研究小组，旨在了解膜蛋白中的变构信号。我打算将瞬态受体电位（TRP）阳离子通道作为模型系统，因为它们表现出迷人的变构行为，对多种生理过程具有重要影响。表征最好的TRP通道之一是TRPV1。有害环境刺激（包括灼烧）激活伤害感受器末端的这一通道，直接导致疼痛的感知，并可能促进炎症性痛觉过敏的发展。所有这些过程最终都依赖于TRPV1孔隙中阳离子传导通路的开启和关闭（即门控），以及正、负调节剂对其的控制。我们目前缺乏对TRP通道门控过程的机制理解，以及通过门控由温度和香草等重要刺激控制的机制。该项目首先旨在确定直接控制孔隙门控的通道区域。其次，它的目的是确定孔的细胞外一半是否形成温度传感器。第三，它将旨在阐明不同香草化合物激活或抑制TRPV1通道的机制。这对于TRPV1靶向镇痛药物对体温调节的影响以及内源性促炎激活剂TRPV1的功能具有重要意义。最后，我将在TRPV2通道中设计对香草素的敏感性，并研究这些分子对通道的调制机制是否在V1和V2之间保守。这三个目标将通过结合通道活性的电生理测量、位点定向诱变、计算生物物理学和结构信息来解决。该奖项的指导部分将使我获得必要的培训，以理解计算生物物理学的方法，并严格地将其应用于我未来的研究。这将补充我作为实验生理学家的训练，为我提供一个扩展的概念框架和一套实用的工具，在原子尺度上的结构动力学水平上研究蛋白质的功能。我认为整合蛋白质的结构、功能和动力学是充分理解控制蛋白质变构的机制所必需的，因此我认为我将获得的培训将是我职业生涯的基础。此外，在斯沃茨实验室（世界上离子通道研究的领先实验室之一）的培训将加强我在电生理学方面的专业知识，拓宽我的整体科学视野。

My long-term goal is to establish an independent research group aimed at understanding allosteric signaling in membrane proteins. I intend to focus on Transient Receptor Potential (TRP) cation channels as a model system, as they exhibit fascinating allosteric behaviors that have important consequences for diverse physiological processes. One of the best characterized TRP channels is the TRPV1. The activation of this channel in nociceptor terminals by noxious environmental stimuli, including burning heat, leads directly to the perception of pain, and can contribute to the development of inflammatory hyperalgesia. All these processes ultimately depend on the opening and closing (i.e. gating) of the cation-conduction pathway in the pore of TRPV1, and its control by positive and negative modulators. We currently lack a mechanistic understanding of the process of TRP channel gating, as well as of the mechanisms through which gating is controlled by important stimuli such as temperature and vanilloids. This project will first aim to identify the regions of the channel that directly control gating of the pore. Second, it will aim at determining whether the extracellular half of the pore forms the temperature sensor. Third, it will aim at elucidating the mechanisms through which distinct vanilloid compounds can either activate or inhibit the TRPV1 channel. This has important consequences for the effects of TRPV1-targetted analgesic drugs on thermoregulation, and for the function of endogenous pro-inflammatory activators of TRPV1. Finally, I will engineer sensitivity to vanilloids into the TRPV2 channel and study whether the mechanisms of channel modulation by these molecules are conserved between V1 and V2. The three aims will be addressed with a combination of electrophysiological measurements of channel activity, site-directed mutagenesis, computational biophysics and structural information. The mentored part of the award will allow me to obtain the necessary training to understand computational biophysics approaches and rigorously apply them to my future research. This will complement my training as an experimental physiologist, providing me with an extended conceptual framework and a set of practical tools to investigate protein function at the level of structural dynamics at atomic scales. I consider that the integration of protein structure, function and dynamics is necessary to fully comprehend the mechanisms that govern allosterism in proteins, and I therefore consider that the training that I will obtain will be fundamental for my career. In addition, training in the Swartz lab, one of the leading laboratories worldwide in ion channel research, will strengthen my expertise in electrophysiology and broaden my overall scientific perspective.