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.
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.
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