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Acid-sensing ion channels (ASICs) are known to be primarily activated by extracellular protons.Recently, we characterized a novel nonproton ligand (2-guanidine-4-methylquinazoline, GMQ) which activates the ASIC3 channel subtype at neutral pH.Using an interactive computational-experimental approach, here we extend our investigation to delineate the architecture of the GMQ sensing domain in the ASIC3 channels.We first established a GMQ-binding mode and revealed that residues E423, E79, L77, R376, Q271, and Q269 play key roles in forming the GMQ sensing domain.We then verified the GMQ-binding mode using ab initio calculation and mutagenesis, and demonstrated the critical role of the above GMQ-binding residues in the interplay among GMQ, proton, and Ca2+ in regulating the function of ASIC3.Additionally, we showed that the same residues involved in coordinating GMQ responses are also critical for activation of the ASIC3E79C mutant by thiol-reactive compound DTNB.Thus, a range of complementary techniques provide independent evidence for the structural details of the GMQ sensing domain at atomic level, laying the foundation for further investigations of endogenous nonproton ligands and gating mechanisms of the ASIC3 channels.