We examined a novel KCNQ2 mutation identified in a Turkish BFNC family predicting the single amino acid substitution N258S located in the linker of S5 to the pore loop. Functional expression in oocytes revealed barely measurable currents of the mutant channel, but preserved formation of heteromeric functional channels when the N258S was expressed with KCNQ3. The N258S exerted a dominant-negative effect on WT currents, which resulted in 40% of current reduction in experiments expressing KCNQ2/N258S/KCNQ3 in a 1:1:2 ratio mimicking a supposed in vivo situation. To unravel a mechanism of the observed loss-of-function of N258S, we constructed fusion proteins comprising mutant or WT KCNQ2 with either KCNQ3 or EGFP and studied them in CHO cells using whole cell and single channel patch clamp recordings and live cell confocal imaging. The current reduction of the KCNQ3/Q2 dimer harbouring the mutant was less pronounced as observed in oocytes and without a dominant negative effect. Single channel parameters were similar for WT and mutant channels and did not reveal a possible mechanism of macroscopic current reduction. Ongoing confocal microscopy studies aim to assess the effects of the mutation on surface expression and targeting.