Structure And Function Relationship Of Acid—Sensing Ion Channels

Acid-sensing ion channels (ASICs), belonging to degenerin/epithelial Na+ channel (DEG/ENaC) superfamily, are non-selective cation channels activated by extracellular protons. In mammals, four asic genes have been found to encode at least 6 ASIC subunits (ASIC1a, lb,2a,2b,3 and 4), which form either homo-or heterotrimeric channels. ASICs are widely expressed in CNS and PNS, and are involved in many physiological and pathological conditions. In PNS, ASICs act as sensory receptors, such as in nociception and mechanosensation. In CNS, ASICs are related with synaptic plasticity, learning, fear conditioning, ischemia and epilepsy. Along with the resolution of crystal structure of cASIC1, the post-crystal era has arrived. In recent years, functional research of ASICs based on crystal structure have emerged prominently. In this research thesis, we have studied structural mechanisms under regulation of expression and gating processes based on the crystal structures of ASICs.As ASICs are activated by extracellular protons, a detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In the first part of our research, we examined the role of a highly conserved salt-bridge residing at the extracellular loop of rat ASIC3 (D107-R153) and human ASIC1a (D107-R160) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt-bridge is essential for functional expression of ASICs in a ligand-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt-bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis and further mutagenesis studies suggested a high stability and structural constrain of the salt-bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt-bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation.Acid-sensing ion channel 3 (ASIC3) channels are extracellular proton (H+)-activated trimeric sodium channels. Generally, proton-induced ASIC3 currents consist of a transient component and a following sustained component. Sustained activation of ASIC3 channels may be responsible for acidosis-associated pain perception and signal integration. Identification of the structural basis for the sustained activation of ASIC3 channels is crucial for the development of pain killers. As the second research part in this thesis, we used FMRF-NH2, a natural peptide which markedly potentiates acid-induced sustained component of ASIC3 channels, as a probe to study the underlying structural mechanisms. In combination with mutagenesis, electrophysiological analysis, chemical synthesis, covalent modification and molecular modeling, we found that the E423-E79-Q269-Q271 (EEQQ) interaction complexes, located in the center of the extracellular region as well as the interface of three ASIC3 subunits, participated in the sustained activation of ASIC3 channels in responses to persistent acidosis. A single mutation or covalent modification of EEQQ complexes conferred the largely sustained activation of ASIC3 channel in responses to persistent acidosis, suggesting an essential role of the EEQQ module. Further molecular dynamic simulations, normal mode analysis and mutation cycle analysis suggested that EEQQ complexes act as a switch that controls the β-linker motif binding or dissociating from thumb domain via an allosteric mechanism, which correlates well with ASIC3 desensitization and sustained activation, respectively. These results elucidate the detailed mechanism for ASIC endogenous regulation and reveal a new conformation transduction pathway for ASIC3 gating, which provide a new structural model and intervention way for drug development and treatment of inflammatory pain.