Redox regulation of Kv7 channels through EF3 hand of calmodulin

Abstract

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. 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 between helices A and B 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 necessary and sufficient to translate Ca2+ signals to reorient the AB fork. Our data is 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 necessary and sufficient for this signaling. SignificanceOxidation-dependent enhancement of the KV7/M-channels plays a cytoprotective role in neurons. Here, we show that calmodulin (CaM), the main protein that conveys information from transient intracellular Ca2+ oscillations, plays a critical role in oxidative signal transduction. The prevailing view is that the main role of the EF-hands is to respond to Ca2+ and that the two EF-hands of CaM in each lobe act in coordination during signaling. We find that EF3 by itself is sufficient and necessary for the oxidative response of Kv7 channel complex and for gating the Calcium Responsive Domain of Kv7 channels. In addition, the direction of EF3-dependent signaling can be reversed by protein-protein interactions with solvent exposed regions outside the target binding groove between EF-hands.