The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external K+. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K+ and Na+ permeation. Permeability ratio measurements, measurements of the lowest K+ required for interaction with the selectivity filter, and measurements of macroscopic K+ and Na+ conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K+ to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K+ permeation properties, Lys 356 appeared to be without effect on Na+ permeation. This suggests that Lys 356 limited K+ flux by interfering with a selective K+ binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K+ permeation properties. Together, these results indicate that a selective, functional K+ binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K+ channels. We suggest that this site is the location of K+ hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K+ current magnitude, but did not influence the ability of internal K+ to enter the pore. These data indicate that in Kv2.1, exit of K+ from the selectivity filter, rather than entry of internal K+ into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K+ channels. PUBLICATION ABSTRACT