Cellular Physiology of Sensory Ion Channels

描述(申请人提供)：化学伤害性感受是初级传入感觉神经元识别有害物质并对其做出反应的过程，导致疼痛、不适和/或神经源性炎症。这类物质包括外源性环境刺激物，以及为应对伤害或疾病而产生或释放的内源性促痛剂。离子通道TRPA1也被称为‘芥末受体’，是化学刺激物的主要探测器，在急性疼痛和慢性炎症性疼痛综合征的潜在机制中起着重要作用，如关节炎、炎症性肠病、出血性膀胱炎、哮喘和其他缩窄性呼吸道疾病。因此，了解TRPA1检测有害化学刺激并对细胞调节机制做出反应的基本机制与基础感觉生理学和疼痛疗法都是相关的。最近的研究表明，化学反应性(亲电性)刺激物通过一种不寻常的过程激活TRPA1，该过程涉及位于通道蛋白胞质氨基末端的所谓连接区域内的半胱氨酸残基的共价修饰。与连接区相邻的Ankyrin-Repeat Rich结构域(ARD)在确定刺激敏感性方面也起着重要作用，这表明它与连接区参与了功能性的、可能的结构上的相互作用，从而介导了刺激诱发的通道门控。细胞内钙 也是TRPA1功能的关键调节器，促进增强和失活。最近的结构-功能研究进一步表明，钙依赖的调节也是由ARD中的一个位点(S)指定的，尽管这一重要调控过程背后的细胞和生化机制仍然不清楚。G蛋白β-γ亚基也可作为TRPA1的调节剂。这项建议的目的是阐明TRPA1氨基末端作为调节感觉神经元兴奋的生理刺激的整合因子的生化和结构机制。具体目标是：(1)界定 连接区的关键结构元素，如长度、与ARD结构域的接近程度和可修饰半胱氨酸残基的相对位置，确定刺激物的检测和通道门控的有效性；(Ii)利用电生理和生化方法阐明G蛋白和钙依赖的通道调节的细胞信号机制；以及(Iii)利用生化和结晶学方法建立TRPA1氨基末端的结构模型。总之，这些研究将为开发和设计控制慢性疼痛和神经源性炎症综合征的新型止痛药提供合理的基础。

DESCRIPTION (provided by applicant): Chemo-nociception is the process whereby primary afferent sensory neurons recognize and respond to noxious substances, resulting in pain, discomfort, and/or neurogenic inflammation. Such substances include exogenous environmental irritants, as well as endogenous pro-algesic agents that are produced or released in response to injury or disease. The ion channel TRPA1 also known as the 'wasabi receptor' functions as a major detector of chemical irritants and contributes significantly to mechanisms underlying acute pain and chronic inflammatory pain syndromes, such as arthritis, inflammatory bowel disease, hemorrhagic cystitis, asthma and other constrictive airway disorders. Thus, understanding basic mechanisms whereby TRPA1 detects noxious chemical stimuli and responds to cellular regulatory mechanisms is relevant to both basic sensory physiology and pain therapeutics. Recent studies have shown that chemically reactive (electrophilic) irritants activate TRPA1 through an unusual process involving covalent modification of cysteine residues within the so-called 'linker region' located in the cytoplasmic amino-terminus of the channel protein. An ankyrin-repeat rich domain (ARD) adjacent to the linker region also plays a significant role in specifying stimulus sensitivity, suggesting that it engages in a functional, and possibly structura interaction with the linker region to mediate irritant-evoked channel gating. Intracellular calcium also serves as a key modulator of TRPA1 function, promoting both potentiation and inactivation. Recent structure-function studies further suggest that calcium-dependent modulation is also specified by a site(s) within the ARD, although the cellular and biochemical mechanisms underlying this important regulatory process remain obscure. G protein beta-gamma subunits may also serve as TRPA1 modulators. The goal of this proposal is to elucidate biochemical and structural mechanisms whereby the TRPA1 amino terminus functions as an integrator of physiological stimuli that regulate sensory neuron excitation. The specific aims are to: (i) define the key structural elements of the linker region - such as length, proximity to the ARD domain, and relative location of modifiable cysteine residues that specify irritant detection and efficienc of channel gating; (ii) elucidate cellular signaling mechanisms underlying G protein and calcium-dependent channel modulation using electrophysiological and biochemical methods; and (iii) develop a structural model of the TRPA1 amino-terminus using biochemical and crystallographic methods. Together, these studies will provide a rational basis for the development and design of novel analgesic agents for controlling chronic pain and neurogenic inflammatory syndromes.