Voltage gating and 4-aminopyridine inhibition in the Shaker Kv channel revealed by a closed-state model.

The generation and propagation of action potentials in neurons relies on the coordinated activation of voltage-dependent sodium and potassium channels. The Kv1 (Shaker) family of potassium channels drives the repolarization phase of the action potential by opening and closing their pore, a process controlled by a voltage sensor domain. However, a molecular description of how the voltage sensor domain drives pore gating has been constrained by a lack of closed-state structures. Here, we present a structural model of the closed Shaker channel that reveals the structural basis of voltage gating. Using AlphaFold2-based conformational sampling, we identified a partially activated state of the voltage sensor which, when modeled with the full channel, produced a closed state. Based on this model we demonstrate that breaking a backbone hydrogen bond between the S4-S5 linker and S5 helices is a critical part of the activation pathway. Docking studies revealed a hydrophobic cavity in the closed pore that binds 4-aminopyridine, a potassium channel inhibitor used to enhance nerve conduction in multiple sclerosis. Our results demonstrate how the voltage sensor movement drives pore opening and provide a structural framework for developing new therapeutic agents targeting the closed state. We anticipate that the novel methods used in this work will allow the characterization of conformational dynamics in voltage-gated ion channels, enabling drug design efforts focused on state-dependent modulation of ion channels for neurological disorders treatment.