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，但是治疗方案有限，并且 当前的阿片类药物流行尤其强调了对新的安全有效治疗的迫切需要。 热TRP通道是最近在周围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药的有吸引力的目标。该提议的目的是完全 表征这些通道中的热传感，并将其与同一蛋白质中的激动剂传感区分开。 当前对通道的研究依赖于斑块夹，该贴片夹有限于检测孔口的检测，而 刺激传感的特性与门控相关，只能从对影响的影响中推断出来 门控。此外，假设贴片响应通常通过缓慢的温度坡道解决，假设 均衡门控最近的研究表明是无效的。这些基本限制会导致不确定性 关于通道的温度敏感性以及机制和诱变的解释。 该应用程序将通过新的量热法扩展研究跨度，以直接探测 通道中的温度传感和独特的激光加热方法，以固定温度激活。 这些新颖的工具将使我们能够分开传感，门控和应对非平衡动力学，从而将 启用对通道的深入机理分析。我们将与这些方法相结合来利用这些方法 脂质体中纯化蛋白的生物物理建模和功能重构，以解决中心 这些通道如何获得强度敏感性的问题。我们的目标1将把量热法利用为 直接检测在重组的TRPV1中热传感的热转变，这是一种典型的热敏感TRP 渠道，并将该测量与贴片记录相结合，以得出完整而严格的分析 通道的热敏感性。 AIM 2将将电生理学和量热法与诱变相结合至 确定是否存在通道蛋白的子域，决定了热感应的能量学和 因此，表现像热传感器。 AIM 3将通过测试来解决多聚TRPV1激活机制 单独使用贴片夹很难区分的几个突出模型。实验将阐明 热敏感性是否位于通道内，从根本上可以与激动剂和其他 刺激。总体而言，应用程序将改变我们研究热通道的能力，发现可以指导 对特定刺激的镇痛药的设计。