In potassium (K+) channels, permeation, selectivity, and gating at the selectivity filter are all governed by the thermodynamics and kinetics of the ion-protein interactions. Specific contacts between the carbonyl groups from the Thr-Val-Gly-Tyr-Gly signature filter sequence and the permeant ions generate four equidistant K+ binding sites, thereby defining the high ion selectivity and controlling the transport rate of K+ channels. Here, we used N-15-labeled ammonium ((NH4+)-N-15) as a proxy for K+ to study ion interaction with the selectivity filter of the prototypical full-length K+ channel KcsA by solution state NMR spectroscopy in order to obtain detailed insights into the physicochemical basis of K+ gating. We found that in the closed inactive state of KcsA (at pH 7) four K+ binding sites are occupied over a wide range of (NH4+)-N-15 concentrations, while in intermediate closed-open conformations (at pH similar to 6) the number and occupancy of K+ binding sites are reduced to two. However, in the presence of the scorpion toxin agitoxin II a total loss of (NH4+)-N-15 binding is observed. (NH4+)-N-15 titration studies allowed us to determine the dissociation constants of the four binding sites with values around 10 mM in the closed state of KcsA. Moreover, kinetic NMR experiments measured in the steady state equilibrium detected an off- and on-rate for (NH4+)-N-15 of ca. 10(2) s(-1) and 10(3) s(-1) between KcsA-bound (NH4+)-N-15 and the bulk. These findings reveal both the thermodynamics and kinetics of the ion binding sites and thus contribute to our understanding of the action of K+ channels.