Mechanisms of Heat Sensing by Nociceptive Vanilloid Receptors

疼痛是人们寻求医疗护理的最常见原因之一，但治疗选择有限， 目前的阿片类药物流行尤其突出了对新的安全有效的治疗方法的迫切需要。 热TRP通道是最近在外周血中发现的一组温度敏感性离子通道， 感觉神经元和角质形成细胞，在那里它们介导热感觉和伤害感受的第一步，以及 已经成为创造新型镇痛剂的有吸引力的目标。该提案的目的是充分 表征这些通道中的热传感，并将其与相同蛋白质中的激动剂传感区分开来。 目前对通道的研究依赖于膜片钳，其仅限于检测孔开放，而 刺激感知的性质，这是变构链接到门控，只能推断出的影响， 门控此外，贴片响应，通常解决缓慢的温度斜坡，分析假设 最近的研究表明平衡门控是无效的。这些基本的限制导致了一种不确定性， 关于通道的温度敏感性以及机制和诱变结果的解释。 这一应用将扩大与量热法直接探测新的研究跨度 通道中的温度传感和独特的激光加热方法，以时间分辨温度激活。 这些新工具将使我们能够分离传感和门控，并解决非平衡动力学问题， 能够对渠道进行深入的机制分析。我们将利用这些方法，结合 脂质体中纯化蛋白质的生物物理建模和功能重建，以解决核心问题。 问题是这些通道如何获得它们的强温度敏感性。我们的目标1将利用量热法， 直接检测重组TRPV1（一种典型的热敏TRP）中的热敏热转变 通道，并将联合收割机的测量与补丁记录，以得出一个完整的和严格的分析 通道的热敏感性。Aim 2将联合收割机电生理学和量热法与诱变相结合， 确定是否存在指示热感测能量学的通道蛋白的亚结构域， 就像热传感器一样。目标3将通过测试来解决多模式TRPV 1激活机制 几个突出的模型是难以区分的膜片钳单独。实验将阐明 热敏感性是否局限于通道内并且从根本上与激动剂和其他激动剂分离， 刺激。总的来说，该应用程序将改变我们研究热通道的能力，研究结果可以指导 对特定刺激具有特异性的镇痛药的设计。

Pain is among the most common reasons people seek medical care, yet the treatment options are limited, and the current opioid epidemic especially highlights the urgent need of new safe and effective therapeutics. Thermal TRP channels are a group of temperature sensitive ion channels recently discovered in peripheral sensory neurons and keratinocytes where they mediate the first step of thermal sensation and nociception, and have emerged as attractive targets for creation of novel analgesics. The goal of this proposal is to fully characterize thermal sensing in these channels and distinguish it from agonist sensing in the same protein. Current studies of the channels rely on patch clamp which is limited to detection of pore opening, while properties of stimulus sensing, which is allosterically linked to gating, can only be inferred from influences on gating. Moreover, patch responses, commonly resolved with slow temperature ramps, are analyzed assuming equilibrium gating that recent studies indicate is not valid. These fundamental limitations cause an uncertainty about temperature sensitivity of channels and interpretations of mechanisms and mutagenesis results. This application will expand the span of the studies with a new methodology of calorimetry to directly probe temperature sensing in channels and a unique laser heating approach to time-resolve temperature activation. These novel tools will allow us to separate sensing and gating and tackle non-equilibrium dynamics and thus enable an in-depth mechanistic analysis of channels. We will exploit these approaches, in conjunction with biophysical modeling and functional reconstitution of purified proteins in liposomes, to address the central question of how these channels obtain their strong temperature sensitivity. Our Aim 1 will exploit calorimetry to directly detect thermal transitions of heat sensing in reconstituted TRPV1, a prototypical heat-sensitive TRP channel, and will combine that measurement with patch recordings to derive a complete and rigorous analysis of heat sensitivity of the channel. Aim 2 will combine electrophysiology and calorimetry with mutagenesis to determine whether there are subdomains of the channel protein that dictate the energetics of heat sensing and are thus acting like heat sensors. Aim 3 will address the polymodal TRPV1 activation mechanisms by testing several prominent models that are difficult to differentiate by patch clamp alone. The experiments will elucidate whether heat sensitivity is localized within the channel and fundamentally separable from agonists and other stimuli. Overall, the application will transform our ability to study thermal channels, and the findings can guide the design of analgesics with specificity to particular stimuli.