TRPM2通道结构与功能及其与新型通道阻断剂相互作用的研究

TRPM2 is a Ca2+-permeable cationic channel gated by intracellular ADP-ribose (ADPR) and, due to its potent activation by reactive oxygen species (ROS), has been shown in recent studies to play an important role in ischemia/reperfusion-induced neurodegeneration and heart infarction, diabetes and inflammatory diseases. Such studies highlight TRPM2 channel inhibition as a promising novel therapeutic strategy. This proposal will study the extracellular pore, or the extracellular vestibule of the transmembrane ion-permeating pore in the human TRPM2 (hTRPM2) channel. The extracellular pore is the key structural module that determines the channel functional properties, and it is also readily accessible from extracellular side; these unique attributes make the extracellular pore as an appealing site for drug targeting.Furthermore, the hTRPM2 extracellular pore is the target of naturally occurring mutation associated with diseases. We aim to delineate the extracellular pore, using three independent and complementary approaches. First, we will compute structural models of the hTRPM2 pore in the closed and open states. The recently available TRPV1 pore structures and strong sequence relatedness in the key pore-forming parts between TRPV1 and TRPM2 provide us an unprecedented opportunity to generate meaningful models of the hTRPM2 pore. Second, using substituted cysteine accessibility and cysteine-based cross-linking methods, we will obtain experimental data to corroborate and refine the structural models of the hTRPM2 extracellular pore. Finally, we will study the interactions of two structurally different TRPM2 open channel blockers with the extracellular pore to identify the interacting residues that are accessible in the open but not closed state. Identification of such extracellular pore residues will provide independent evidence to elaborate the structural models of the hTRPM2 extracellular pore. The structure-function relationship information of the hTRPM2 extracellular pore gleaned from this project will enable us to gain a mechanistic insight into TRPM2 channel activation, offer a better understanding of the disease mechanisms and provide highly desirable knowledge for the expanding efforts of developing small molecular chemicals as TRPM2 channel blockers for therapeutic use.

TRPM2是ADPR门控瞬时受体电位通道,可被活性氧激活,被认作是一种新型氧化应激感受器。利用基因敲除小鼠,揭示TRPM2在缺血再灌注损伤、神经退行性病变、炎症等过程中发挥关键作用；因此 TRPM2作为新型药物靶点已引起国际制药行业极大兴趣。TRPM2通道是同源四聚物，每个亚基包含6个跨膜片段（S1-S6）和S5-S6之间的P环。P环和S5、S6胞外端 构成通道胞外孔区。胞外孔区既是决定通道特殊功能的关键结构区域，又是药物最易结合的位点。目前对TRPM2胞外孔区知之甚少。本研究利用刚解析的TRPV1通道结构及其与TRPM2氨基酸序列的保守性构建TRPM2通道结构模型。结合定点突变和交联技术与膜片钳技术验证胞外孔区结构模型，并通过解析两种不同的新型通道阻断剂的结合位点来确认和完善TRPM2胞外孔区结构。阐明TRPM2通道结构和功能关系为理解其生理功能、相关发病机制和药物研制所需信息奠定基础。

TRPM2是ADP-ribose门控瞬时受体电位通道, 可被活性氧激活, 被认作是一种新型氧化应激感受器。本研究中，（1）我们研究TRPM2通道胞外孔区，该区是决定该通道特殊功能的关键结构区域。利用TRPV1通道结构及其与TRPM2通道形成孔区的氨基酸序列的保守性，构建了TRPM2通道孔区结构模型，然后结合定点突变与膜片钳技术，解析两种不同的新型通道阻断剂（ACA和cmpd-39）的结合位点，确认和完善了TRPM2通道胞外孔区的结构和功能关系；（2）我们结合定点突变与膜片钳技术，研究配体ADPR/cADPR与TRPM2通道C-末端NUDT9-H区域作用，解析出与配体结合的关键氨基酸残基从而阐述激活TRPM2 通道的关键步骤。 这些结果帮助理解TRPM2的激活机制，为设计新型和特异性TRPM2通道拮抗剂提供所需的结构与功能关系。另外，我们利用转基因TRPM2通道敲除小鼠研究TRPM2通道在脑损伤与疾病中的作用，揭示：（1）TRPM2通道介导缺血再灌注引起的神经元胞内锌离子的升高从而破坏动态平衡，引起神经元死亡，导致脑损伤；（2）TRPM2通道介道氧化应激引起的足细胞死亡从而导致血脑屏障损伤；（3）粉样蛋白引起海马区神经元TRPM2通道激活，从而导致溶酶体与线粒体功能破坏，直接引起神经元死亡；（4）TRPM2通道介导老年痴呆症相关的神经炎症；TRPM2 通道介导淀粉样蛋白引起小胶质细胞激活和释放肿瘤坏死因子-α，进而激活神经元细胞表面肿瘤坏死因子受体，间接引起神经元死亡。这些结果揭示TRPM2 通道在缺血再灌注脑损伤、血脑屏障损伤 、神经退行性病变等过程中的起到关键作用。我们提出TRPM2作为治疗脑疾病与保护脑功能损伤的新型药物靶点。我们的研究已引起国际同行的关注。