Benign familial neonatal convulsions (BFNC) is an autosomal dominant epileptic disorder resulting from mutations in two voltage-gated potassium channel genes, KCNQ2 and KCNQ3 (channels K_V7.2 and K_V7.3). We identified a novel KCNQ2 mutation in a BFNC family with three affected individuals, which predicts the amino acid substitution p.S122L located in the S2 segment of the voltage-dependent K_V7.2 channel. The S122L mutation was functionally expressed in Xenopus laevis oocytes and analyzed using two-microelectrode voltage clamping. Comparison of the biophysical properties of the mutant S122L channel with wildtype (WT) K_V7.2 revealed a significant positive shift in the voltage dependence of activation with an increased slope and a slowing of the activation time course. The shift was most pronounced in the subthreshold range of an action potential predicting a current reduction of 75% at -50 mV, which is likely to be the physiologically relevant voltage range for this non-inactivating potassium channel to regulate neuronal firing. These results establish an important role of the K_V7.2 S2 segment in voltage-dependent channel gating and suggest that subtle mutation-induced gating alterations restricted to the physiologically relevant subthreshold range can cause neonatal epilepsy.