The Isolated Thumb Domain of Acid-Sensing Ion Channels Forms a Minimal Folding Unit Enabling Ligand Binding Studies

Proton-gated acid-sensing ion channels (ASICs) are emerging therapeutic targets for ischemia-related conditions such as stroke and myocardial infarction. Although structural data exist for ASIC1a, key aspects of ligand recognition and modulation remain unresolved. Using multidimensional solution-state NMR spectroscopy, we show that the principal ligand-binding region of ASICs, the thumb domain, forms an independently folded unit that at neutral pH adopts a native-like conformation resembling the resting state of the channel. By integrating high-resolution biophysical analyses of ligand binding to the isolated thumb domain with electrophysiological measurements on full-length ASIC1a, we distinguished molecular interactions that determine binding affinity from those governing functional efficacy. This approach revealed that dynorphin A acts as a competitive antagonist of ASIC1a. Furthermore, NMR-based pK_a determination of individual acidic residues demonstrated generally elevated values across the isolated thumb domain, supporting the presence of an extended acid-sensing network rather than a single dominant pH sensor. These findings establish the isolated thumb domain as a powerful model for dissecting ASIC ligand interactions and pH sensitivity in solution, providing mechanistic insights and enabling structure-based drug discovery of therapeutic modulators for ASICs and related ion channels.