Ion flow in many voltage-gated K channels (VGK), includ - ing the (human ether-a-go-go -related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approxi- mately every third residue, from Phe 551 to Trp 563 , affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on acti- vation (W563A, F559A, and F551A) face out toward the volt- age sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala 561 , His 562 , Ala 565 , Trp 568 , and Ile 571 ), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.