Mechanisms of Heat Sensing by Nociceptive Vanilloid Receptors

伤害性香草素受体的热感应机制

• 批准号: 9973924
• 负责人: FENG QIN
• 金额: $ 39.07万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973924
• 关键词: Address; Affect; Afferent Neurons; Agonist; American; Analgesics; Ankyrin Repeat; Calorimetry; Caring; Collection; Coupling; Detection; Development; Diabetes Mellitus; Differential Scanning Calorimetry; Discrimination; Electrophysiology (science); Environment; Equilibrium; Esthesia; Exhibits; Goals; Heart Diseases; Heating; High temperature of physical object; Homologous Gene; Hyperalgesia; Ion Channel; Ion Channel Gating; Kinetics; Knowledge; Lasers; Link; Liposomes; Malignant Neoplasms; Measurement; Mediating; Medical; Medicine; Membrane Fluidity; Methodology; Modality; Modeling; Molecular; Mutagenesis; Nociception; Pain; Pathway interactions; Peripheral; Property; Proteins; Ramp; Site; Specificity; Stimulus; TRP channel; TRPV1 gene; Temperature; Temperature Sense; Testing; Therapeutic Intervention; Thermodynamics; Time; Treatment Efficacy; Uncertainty; Variant; biophysical model; capsaicin receptor; cofactor; design; enthalpy; experimental study; field study; improved; keratinocyte; novel; opioid epidemic; pain relief; pain signal; patch clamp; reconstitution; response; sensor; side effect; theories; therapeutic target; tool; voltage

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.

疼痛是人们寻求医疗服务的最常见原因之一，但治疗选择有限，而且 目前的阿片类药物流行特别突出了迫切需要新的安全有效的治疗方法。 热Trp通道是最近在外周发现的一组温度敏感离子通道 感觉神经元和角质形成细胞，它们介导热感觉和伤害性感觉的第一步，以及 已经成为创造新型止痛药的有吸引力的目标。这项提议的目标是充分 描述这些通道中的热感觉，并将其与同一蛋白质中的激动剂感觉区分开来。 目前对通道的研究依赖于膜片钳，这仅限于检测孔道开放，而 与门控变构相关的刺激传感的特性只能从对 门控。此外，通常用缓慢的温度梯度解决的补丁响应被分析，假设 最近的研究表明，均衡门控是不成立的。这些根本性的限制导致了不确定性 关于通道的温度敏感性以及对机制和诱变结果的解释。 这一应用将扩大研究的跨度，以一种新的量热方法直接探索 通道中的温度传感和独特的激光加热方法可对温度激活进行时间分辨。 这些新的工具将允许我们分离感知和门控，并处理非平衡动态，从而 实现对渠道的深入机制分析。我们将利用这些方法，与 脂质体中纯化蛋白的生物物理模拟和功能重建，以解决中央 这些通道如何获得其强烈的温度敏感性的问题。我们的目标1将利用量热技术来 直接探测重组TRPV1热敏Trp的热敏转变 频道，并将把该测量与补丁记录相结合，以得出完整而严格的分析 通道的热敏感度。目标2将电生理和量热与诱变相结合，以 确定是否存在通道蛋白的亚域来决定热敏和 因此它们的行为就像是热传感器。目标3将通过测试解决多模式TRPV1激活机制 仅靠膜片钳很难区分的几个突出的模型。这些实验将阐明 热敏性是否局限于通道内，并从根本上与激动剂和其他 刺激物。总体而言，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止痛药的设计。