Potassium channels selectively conduct K+ ions across cell membranes, and use diverse mechanisms to control their gating. We studied ion permeation and gating of an inwardly rectifying K+ channel by individually changing the amide carbonyls of two conserved glycines lining the selectivity filter to ester carbonyls using nonsense suppression. Surprisingly, these backbone mutations do not significantly alter ion selectivity. However, they dramatically change the kinetics of single-channel gating and produce distinct subconductance levels. The mutation at the glycine closer to the inner mouth of the pore also abolishes high-affinity binding of Ba2+ to the channel, indicating the importance of this position in ion stabilization in the selectivity filter. Our results demonstrate that K+ ion selectivity can be retained even with significant reduction of electronegativity in the selectivity filter, and that conformational changes of the filter arising from interactions between permeant ions and the backbone carbonyls contribute directly to channel gating.