Knowledge Bank

Acid Sensing Ion Channels (ASICs) are cation channels in the central nervous system that are activated by acidic pH. ASICs are utilized in normal physiological processes, such as learning and memory and pain. They have also been implicated in various neurological disorders, including ischemic stroke and traumatic brain injury. These neurological disorders induce acidosis, with the resulting ASIC activation initiating a cascade of events leading to cell death. The details of how this pathway occurs are not well known, but newer research suggests that it occurs through ASIC's protein-protein interactions. The purpose of this study is to develop a method to identify the proteins and pathways which contribute to ASIC-dependent effects. Proximity labeling is an unbiased approach of elucidating the ASIC1a-induced acidotoxicity pathway. We investigated two forms of proximity labeling, biotin ligase BioID2 and engineering peroxidase APEX2. These promiscuous enzymes attached biotin to neighboring proteins within a certain radius of the target protein. We employed two constructs, with BioID2 and APEX2 being fused to the C-terminus of ASIC1a. APEX2 was selected as it is a quicker form of proximity labeling that allows for tagging of protein-protein interactions with ASICs under acidosis conditions. When exposed to hydrogen peroxide, the APEX2 system oxidizes biotinyl tiramide to create radicals which allow biotinylation of proteins. The biotinylated proteins are then isolated via Streptavidin beads. Through this methodology, we have been able to isolate biotinylated proteins and are currently using this system to identify acid-dependent protein interactions of ASICs through proteomics. By elucidating the proteins interacting with ASICs, we will lend further insight into the signaling cascades that lead to cell death, which may yield novel therapeutic strategies to aid in the treatment of multiple neurological diseases.