The acid, sensing joh channel (ASIC) is a member of the epithelial sodium channel/degenerin family of sodium ion channels. Within the ASICs, there is strong homplpgy between proteins. The ASIC receptor can detect changes in pH within a physiolgically relevant range, making the receptor useful in detecting muscle ischemia or stroke-induced ischemia. Developing specific antagonists for ASIC receptors may enhance current treatment of pain or preventing neurotoxicity. The physiology of the ASIC receptor is beginning to be understood. The channel gating mechanism and the location of agonist proton-binding sites are unresolved. Recently the Gpuaux laboratory determined the crystal structure of a non-functional chicken ASIC1 construct to 1.9 angstrom resolution. Using this structure as a guide, I will address two fundamental questions that govern ASJC receptor activity: 1) what is the structure of a functipnal ASIC receptor, and 2) where are the agpnjst prptpn-binding sites that induce channel gating. To address the functional ASIC construct, I wilj systematically replace sections of N- and C-terrninj to the truncated construct to identify a minimaj fimctjpn construct. I will concurrently assess the functionality pf the channel (patch clamp electrophysipiogy) and monodispersity (fluorescence-detection size-exclusion chromatpgraphy). Once suitable constructs are identified, the structure will be determined for closed and open states pf the channel. The second goal is to identify and characterize potential agonist protpn-binding sites in ASIC receptors. We have developed criteria fpr defining agonist-binding residues: 1) acidic residues, 2) 3.5 A frpm another acidic residue, and 3) alter cASId proton sensitivity arid/or channel gating. These residues will be mutated individually to variants that mimic proton 'binding'. All mutations that affect channel gating will be incorporated jntp a single multiple mutant receptor. Finally, I will determine the crystal structure using my mipimal function construct of selected mutations that altered channel activation gating. Relevance: Information obtained from the chicken ASIC1 receptor can be applied in the study pf Pther acid sensing ion channels. Our proposed crystallographic studies will aid our understanding of ASICs and lead to development of novel therapeurtics in treating pain and preventing neurotoxicity. ? ? ?
| Gonzales, Eric B; Kawate, Toshimitsu; Gouaux, Eric (2009) Pore architecture and ion sites in acid-sensing ion channels and P2X receptors. Nature 460:599-604 |
