Redox regulation of K_V7 channels through EF3 hand of calmodulin

Neuronal K_V7 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 K_V7.4 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 K_V7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.