The bacterial potassium (K(+)) channel KcsA provides an attractive model system to study ion permeation behavior in a selective K(+)-channel. We changed residue at the N-terminal end of the selectivity filter of KcsA (T74V) to its counterpart in inwardly rectifying K(+)-channels (Kir). The tetramer was found to be stable as unmodified KcsA. Under symmetrical and asymmetrical conditions, Na(+) increased the inward current in the virtual absence of K(+) however outward currents were nearly abolished which could be recovered upon internal K(+) addition. Na(+) also drastically increased the channel open time either in the presence or virtual absence of K(+). Furthermore, the T74V mutation decreased the internal Ba(2+) affinity of the channel possibly by binding to a K(+) site in the pore. In additional experiments, another point mutation V76I in T74V mutant was carried out thus the selectivity filter resembled more the selectivity filter of Kir channels. The mutant tetramer was converted into monomers as determined by conventional gel electrophoresis. However, native like gel electrophoresis, Trp fluorescence and acrylamide quenching experiments indicated that this mutant still formed a tetramer and apparently adopted similar folding properties as unmodified KcsA. Single-channel experiments further demonstrated that the channel was selective for K(+) over Na(+) as Na(+) blocked channel currents. These data suggest that single point mutation T74V alters the selectivity filter and allows simultaneous occupancy and conduction of K(+) and Na(+) probably via ion-ion interaction in the pore. In contrast, both mutations (T74V and V76I) in the same molecule seem to reorganize the pore conformation which controls the overall stability of a selective K(+)-channel.