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Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of KV7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves KV7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling. Ministerio de Ciencia e Innovacion PID2021-128286NB-100Wellcome Trust 212302/Z/18/ZMedical Research Centre MR/P015727/1Eusko Jaurlaritza IT1707-22 Ekonomiaren Garapen eta Lehiakortasun Saila, Eusko Jaurlaritza BG2019Ministerio de Ciencia e Innovacion RTI2018-097839-B-100Ministerio de Ciencia e Innovacion RTI2018-101269-B-I00Eusko Jaurlaritza IT1165-19 Ekonomiaren Garapen eta Lehiakortasun Saila,Eusko Jaurlaritza KK-2020/00110Eusko Jaurlaritza PRE_2018-2_0082Eusko Jaurlaritza POS_2021_1_0017Eusko Jaurlaritza PRE_2018-2_0126