Gating consequences of charge neutralization of arginine residues in the S-4 segment of K(v)7.2, an epilepsy-linked K+ channel subunit

The K(v)7.2 subunits are the main molecular determinants of the M-current, a widespread K+ current regulating neuronal excitability. Mutations in the K(v)7.2 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 S-4 segment of K(v)7.2. In this study, each of the six S-4 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 S-4 arginine residue plays a relevant role in the voltage-dependent gating of K(v)7.2 channels. In particular, a decreased positive charge at the N-terminal end of S-4 stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S-4 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.