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Acid-sensing ion channel 3 (ASIC3) channels are extraeellular proton (H+)-aetivated 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.In this study, we used FMRF-amide, a natural peptide which markedly potentates 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 mutation cycle analysis, and molecular modeling, we found that the 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 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.Thus, we identify EEQQ module as the structural machinery supporting the sustained activation of ASIC3 channels in responses to persistent acidosis.