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具有挑战性，因此治疗策略的发展也相应受到阻碍。最近的研究主要集中在Na+选择性酸敏感离子通道1a （ASIC1a）上，这些研究说明了特定的ASIC1a抑制剂，一种蜘蛛毒素，psalmotoxin1的神经保护作用。针对ASIC1a预防神经元损伤的策略有希望，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，它是如何打开和关闭的，或门是不可或缺的。当暴露于低pH时，通道构象发生改变，打开一个允许离子通过的孔。然而，在质子持续存在的情况下，通道不会保持开放，而是进入一种被称为脱敏状态的非导电状态。我推测脱敏是由于在细胞外区域的亚基界面触发的结构运动，破坏了跨膜区域的开放孔的稳定性。最近，鸡ASIC1a （cASIC1a）的低pH晶体结构被确定为脱敏状态。因此，为了进一步了解通道门是如何形成的，以及它是如何从打开状态转变为脱敏状态的，我将确定cASIC1a在打开状态下的晶体结构。本研究描述了稳定cASIC1a开放状态的不同策略，并通过x射线晶体学确定其结构。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。我将用于拟议研究的方法是x射线晶体学，电生理学和闪烁接近试验。

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. PUBLIC HEALTH RELEVANCE: The current strategy for the treatment of stroke demonstrates effective results only when applied within three hours after onset of stroke. Mounting evidence has shown the involvement of acid-sensing ion channel 1a (ASIC1a) in neuronal injury that results from stroke. Studying ASIC1a at the atomic level will advance our knowledge in the mechanism by which ASIC1a functions, thus, contributing to the development of therapeutic strategies for stroke.