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。我打算将重点放在瞬时受体电位(Trp)阳离子通道作为一个模型系统，因为它们表现出迷人的变构行为，对不同的生理过程具有重要的影响。TRPV1是最具特性的Trp通道之一。伤害性环境刺激，包括灼热，激活伤害性感受器终端的这一通道，直接导致痛觉，并可促进炎症性痛觉过敏的发展。所有这些过程最终依赖于TRPV1孔内阳离子传导通路的开启和关闭(即门控)，以及正、负调节因子的控制。我们目前缺乏对Trp通道门控过程的机械理解，也缺乏对门控受重要刺激如温度和香草素控制的机制的了解。该项目将首先确定通道中直接控制气孔门控的区域。其次，它的目标是确定毛孔的细胞外半部分是否形成温度传感器。第三，它将致力于阐明不同的香草类化合物激活或抑制TRPV1通道的机制。这对于TRPV1靶向止痛药对体温调节的影响以及对TRPV1的内源性促炎激活剂的作用具有重要的影响。最后，我将把对香草素的敏感性引入TRPV2通道，并研究这些分子对通道的调制机制是否在V1和V2之间是保守的。这三个目标将结合通道活性的电生理测量、定点突变、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，并将它们严格应用于我未来的研究。这将补充我作为一名实验生理学家的培训，为我提供一个扩展的概念框架和一套实用工具，以在原子尺度的结构动力学水平上研究蛋白质功能。我认为蛋白质结构、功能和动力学的整合对于充分理解蛋白质变构作用的机制是必要的，因此我认为我将获得的培训将是我职业生涯的基础。此外，在世界领先的离子通道研究实验室之一的斯沃茨实验室接受培训，将加强我在电生理学方面的专业知识，并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.