The second tryptophan (W) residue of the conserved WW motif in the pore helix of many K+ channel subunit is thought to interact with the tyrosine (Y) residues of the selectivity filter. A missense mutation causing the replacement of the corresponding residues with an arginine (W309R) occurs in KCNQ3 subunits forming part of M-channels. In this study, we examined the functional consequences of the W309R mutation in heterogously expressed KCNQ channels. Homomeric KCNQ3W309R channels lacked KCNQ currents. Heteromeric KCNQ2/KCNQ3W309R channels displayed a dominant-negative suppression of current and a significant modification in gating properties when compared with heteromeric KCNQ3/KCNQ2 channels mimicking the M-channels. A three-dimensional homology model in the W309R mutant indicated that the R side chain of pore helices is too far from the Y side chain of the selectivity filter to interact via hydrogen bonds with each other and stabilize the pore structure. Collectively, the present results suggest that the second W residues of pore helices and their chemical interaction with the Y residues of the selectivity filter are essential for normal K+ channel function. This pore-helix mutation, if occurs in the brain M channels, could thus lead to a channel dysfunction sufficient to trigger epileptic hyperexcitability.