In K+ channels, C-type inactivation locks the selectivity filter in a non-conductive state. The prokaryotic proton-gated potassium channel KcsA undergoes a time dependent inactivation process. The interaction between Glu71 and Asp80 is one of the key driving forces that promote filter instability and inactivation. This interaction promotes a compression of the selectivity filter parallel to the permeation pathway, which biases it towards the inactivated conformation. Since KcsA represents a structural model for the pore domain of K+ channels, it is obvious that a detailed understanding of the molecular basis of inactivation is not limited to this prokaryotic channel, but offers new directions into how inactivation gating might proceed in other K+ channels. Here, using patch clamp experiments, EPR spectroscopy, functional assays, molecular dynamics, and X-ray crystallography we show that interactions involving residues Trp67, Tyr78, and Asp80 in KcsA, conserved in most potassium channels, also constitute critical contacts between the selectivity filter and its adjacent pore helix which determines the rate and extent of C-type inactivation in Shaker, Kv1.2, and hERG. Substitution of a tryptophan or tyrosine at the pore helix to phenylalanine in these channels decreases the rate and extent of inactivation, pointing this position as key modulator of gating. Furthermore, by substituting equivalent amino acids critical for hERG inactivation in KcsA we were able to create a non-conducting KcsA mutant with normal pH activated lower gate. These results suggest commonalities in inactivation gating mechanism of eukaryotic channels, and provide evidence that the hydrogen bond network and Van der Waals interactions between the pore helix, selectivity filter, and external vestibule serve as the basis for C-type inactivation in the K+ channel family.