Voltage-gated potassium (Kv) channels play an essential role in cellular excitability and the propagation of nerve impulses. Despite tremendous advances in structural determination, achieving a fundamental, atomic-level understanding of ion channel function—encompassing permeation, selectivity, activation, inactivation, and regulation—remains a formidable biophysical challenge. Fast and selective ion conduction through K⁺ channels is governed by a delicate balance of strong electrostatic interactions among partially dehydrated ions translocating along a narrow pore. Voltage activation of Kv channels involves large and complex conformational rearrangements mediated by the voltage-sensing domain (VSD), which opens the intracellular gate of the channel in response to changes in the transmembrane potential. C-type inactivation arises from conformational alterations of the pore that drive it toward a non-conducting state. Classical molecular dynamics (MD) simulations based on accurate atomic models provide a powerful framework for elucidating the mechanisms underlying these complex biomolecular systems. Here, we examine and delineate the microscopic conditions governing classical multi-ion conduction via the “knock-on” mechanism in K⁺ channels. We find that the occupancy of the narrow pore by ions and water molecules is extremely sensitive to small (~kBT) variations in the interaction between ion, water, and backbone carbonyls. To probe the electromechanical coupling mechanism, we used single-particle cryo-EM to determine the structure of the ILT mutant of the voltage-gated Shaker channel known to exhibit a long-lived closed intermediate state during activation. The results, integrating experimental structure together with computational modeling and MD simulations, provide unprecedented mechanistic insight into how structural rearrangements underpin voltage activation in Kv channels. Lastly, to investigate the molecular basis of C-type inactivation, simulations were performed based on the high-resolution structure of a strongly inactivated triple-mutant channel of Kv1.2–Kv2.1–3, which revealed a novel conformation of the selectivity filter dilated at its outer end—distinct from the well-characterized conductive state. Remarkably, the dilated filter is found to be conductive, and a secondary gate is required to block ionic current.
