3D Structure of Acid-sensing Ion Channel in the Open State

描述(由申请人提供)：建立缺血性神经元损伤的分子机制一直是具有挑战性的，因此治疗策略的发展也相应地受到阻碍。最近的研究集中在Na+选择性酸敏离子通道1a(ASIC1a)上，研究表明了特定的ASIC1a抑制剂，蜘蛛毒素1，一种蜘蛛毒素的神经保护作用。针对ASIC1a的预防神经元损伤的策略是有希望的，但需要在分子水平上对ASIC1a离子通道有广泛的了解。ASIC1a的结构知识对于理解它的功能、它如何打开和关闭或门是不可或缺的。一旦暴露在低pH值下，通道构象就会改变，打开一个允许离子通过的孔洞。然而，在质子持续存在的情况下，该通道并不保持打开，而是进入一种称为脱敏状态的非导电状态。我推测，脱敏是由于细胞外区亚基界面上的结构运动，破坏了跨膜区开放的孔的稳定性。最近，代表脱敏状态的同质鸡ASIC1a(CASIC1a)的低pH晶体结构被确定。因此，为了进一步了解通道门如何以及它如何从开放状态转换到不敏感状态，我将确定cASIC1a在开放状态下的晶体结构。拟议的研究描述了稳定cASIC1a的开放状态并通过X射线结晶学确定其结构的不同策略。这些策略包括形成具有高度特异性和强效毒素的ASIC1a复合体，引入突变，以及添加开放通道阻滞剂。我将使用的研究方法是X射线结晶学、电生理学和闪烁邻近分析。

DESCRIPTION (provided by applicant): Establishing molecular mechanisms underlying ischemic neuronal injury has been challenging and thus the development of therapeutic strategies has been correspondingly hampered. Recent research has focused on the Na+selective acid-sensing ion channel 1a (ASIC1a) based on studies illustrating the neuroprotective effect of the specific ASIC1a inhibitor, psalmotoxin1, a spider toxin. Strategies targeting ASIC1a to prevent neuronal injury hold promise but require extensive knowledge of the ASIC1a ion channel at the molecular level. Structural knowledge of ASIC1a is integral to understanding its functions, how it opens and closes, or gates. Upon exposure to low pH, the channel conformation is changed, opening a pore that allows ions to pass through. The channel does not remain open in the continued presence of protons, however, but rather it proceeds to a non-conducting state known as the desensitized state. I speculate that desensitization is due to structural movements triggered at the subunit interface in the extracellular domain, destabilizing the open pore in the transmembrane domain. Recently, a low pH crystal structure of a homomeric chicken ASIC1a (cASIC1a) representing the desensitized state was determined. Therefore, to further our understanding of how the channel gates and how it transitions from an open to a desensitized state, I will determine the crystal structure of cASIC1a in the open state. The proposed research describes different strategies for stabilizing the open state of cASIC1a and determining its structure by X-ray crystallography. These strategies include forming a complex of the ASIC1a with a highly specific and potent toxin, introduction of mutations, and addition of open channel blockers. Methods I will use for the proposed research are X-ray crystallography, electrophysiology, and scintillation proximity assay.