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

ABSTRACT 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 p K 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.