Acid-Sensing Ion Channel gating: Conformations and Consequences

PROJECT SUMMARY/ABSTRACT Extracellular pH is a highly dynamic and ubiquitous signal and many cell types exhibit robust electrophysiological responses upon extracellular acidification, particularly in the nervous system. Acid-sensing ion channels (ASICs) are thought to mediate the majority of these responses since various ASIC subunits are expressed at high levels in many neuronal types and genetic ablation of ASIC subunits dramatically reduces acid-evoked responses. Consequently, ASICs are vital players in numerous physiological and pathophysiological circumstances including ischemic stroke, basal neurotransmission, synaptic plasticity, pain, inflammation and various cancers. These myriad roles of ASICs have motivated structural and biophysical investigation, leading to crystal or cryo-EM structures of chicken ASIC1 in the resting, toxin-stabilized open and desensitized states. Combining these structural efforts with functional experiments have led to a working model where protonation of key acidic residues in the extracellular domain's acidic pocket leads to global rearrangements driving activation and desensitization. In recent years we have made key insights into the molecular determinants of the activation and desensitization processes at individual subunits. Yet we lack a clear understanding of how these subunits work together. Are conformational changes at one subunit sufficient to desensitize ASICs? Does conformational change in one subunit co-operatively influence the adjacent subunits? We will address these core questions by measuring functional properties of channels with defined stoichiometry. In addition, we will employ powerful next-generation fluorometry approaches and single molecule FRET studies. Finally, to gain insight into the pH stimulus encountered by ASICs we will use protein engineering to measure physiological pH changes. Taken together, these proposed experiments will provide insight into essential unanswered questions in ASIC biology while developing novel tools of broad applicability.