The Kv7.2 subunits are the main molecular determinants of the M-current, a widespread K+ current regulating neuronal excitability. Mutations in the Kv7.2 gene cause benign familial neonatal seizures, an autosomally inherited human epilepsy. The benign familial neonatal seizure-causing mutations include those at arginine residues at positions 207 and 214 in the S4 segment of Kv7.2. In this study, each of the six S4 arginines was individually replaced with neutral glutamines, and the functional properties of mutant channels were studied by whole-cell and single-channel voltage-clamp measurements. The results obtained suggest that each S4 arginine residue plays a relevant role in the voltage-dependent gating of Kv7.2 channels. In particular, a decreased positive charge at the N-terminal end of S4 stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S4 favored the resting conformation. Strikingly, neutralization of a single arginine at position 201 was sufficient to cause a significant loss of voltage dependence in channel activation. Moreover, by comparing the functional properties of glutamine versus tryptophan substitution, we found steric bulk to play a relevant role at position 207, but not at position 214, in which the main functional effect of this disease-causing mutation seems to be a consequence of the loss of the positive charge.
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