The voltage-gated potassium channels K_v7.2 and K_v7.3 (KCNQ2/3 genes) contribute to the neuronal M-current and play an important role in regulating neuronal firing. Mutations in different regions of this gene are linked to benign familial neonatal seizures (BFNS), an inherited form of epilepsy. Recent results showed that a highly conserved Threonine (Thr) residue in the S1-S2 loop is crucial for channel function, in particular for trafficking of different voltage-gated potassium channels to the surface membrane. In K_v7.2, two mutations in the S1-S2 region, S122L and E119G, have been linked to epilepsy without effects on trafficking but subtle changes of voltage-dependent activation. We were interested about the role of the homologous Thr residues in K_v7.2 and K_v7.3 (Q2-T114/Q3-T144) which are conserved in these channels as well. Wild-type (wt) and mutant channels, in which the respective Thr were replaced by Ala, were expressed in mammalian CHO cells and K⁺ currents were analyzed using whole cell patch-clamping. Mutant channels showed significantly reduced current amplitudes and slowed activation kinetics. In addition, we detected a depolarizing shift in the voltage dependence of activation of up to 35 mV in heteromeric mutant Q2-T114A/Q3-T144A channels compared to wt channels. The effects were more pronounced for Q2-T114A than for Q3T144A. These results indicate that the Thr residues in S1-S2 loops play an important functional role in K_v7 channels, mainly affecting voltage-dependent gating and less trafficking.