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We have known the structure for the closed-state of a potassium channel pore domain (PD) for more than a decade. However, major progress in understanding the molecular basis for activation and inactivation gating in K-channels had to wait until high-resolution structural information of the channel in the open state became available. Recently, we solved the structure KcsA in it fully open conformation, as well four others partial openings, which richly illustrated the channel activation-inactivation pathway. Analysis of these open structures suggested that residue F103 in TM2 interacts with the c-terminal end of the pore helix, compressing the pitch of its first helical turn. As a consequence, the distance between E71-D80 side chains is shortened, strengthening the carboxyl-carboxylate interaction that leads to a non-conductive conformation of the selectivity filter. Perturbation mutagenesis at position 103, affected gating kinetics as predicted from our structural analysis: small side chain substitutions F103A and F103C severely impaired inactivation kinetics, suggesting an allosteric coupling between the inner helical bundle and the selectivity filter. Free energy calculations show strong open state interaction-energies between F103 and surrounding residues. Similar interactions were probed in the Shaker K-channel by mutating highly conserved I470, equivalent to F103, to a smaller side chain. In the mutant I470A, inactivation was abrogated, suggesting that a similar mechanism underlies inactivation coupling in eukaryotic potassium channels. A crystallography study of these mutants in the open KcsA will be reported.

The potassium ion channel, KcsA, gates the passage of ions through cell membranes in response to a change in pH. Recent experimental results have demonstrated the existence of two gates in KcsA: an intracellular gate and a gate at the selectivity filter. Lowering the pH opens the intracellular gate allowing ions to pass. After a period of time, however, the channel inactivates by constricting the selectivity filter and impeding the flow of ions even though the bottom gate remains open. We have used path-based molecular dynamics simulations to probe the detailed mechanism of this phenomenon by finding dynamical pathways to inactivation in KcsA. We have computed free energies and rates of inactivation that agree with recent experimental results. We also provide a molecular rationalization for the coupling between the opening and closing of the lower gate and the inactivation of the selectivity filter.