The Closed State of the Shaker Potassium Channel and the Mechanism of Voltage Activation

Voltage-gated potassium (Kv) channels play a critical role in cellular excitability and the propagation of the nerve impulse. Despite the extensive amount of information available about Kv channels, the complete process of voltage activation-where a conformational change mediated by voltage-sensor domains (VSD) opens the intracellular gate in response to the depolarization of the membrane potential-has not been fully elucidated. To understand this process, we study the Kv Shaker channel and focus on the ILT mutant (V369I, I372L, S376T) known to display a long-lived closed intermediate state during the activation process. Single particle CryoEM of the ILT mutant reveals a novel conformation of Shaker in which the intracellular gate formed by the S5-S6 helices is in a closed state and the S4 helix in the VSD is in an intermediate state shifted down by ~5Å relative to its position in the fully activated open channel. Additional conformations of the channel generated by Alphafold2 and molecular dynamics simulations are used to map the sequence of intermediate states along the voltage activation process, revealing the nature of the couping between the VSD, the S4-S5 linker, and the S5-S6 intracellular gate. In the final stage of the activation process, as the VSD reaches its fully activated conformation while the gate remains closed, essential interactions between the S4-S5 linker and the intracellular S5-S6 gate are transiently disrupted and reformed when the gate opens, demonstrating that electro-mechanical coupling results from a dynamic shift in population equilibrium between metastable states. These findings provide unprecedented mechanistic insight into how structural rearrangements underlie the voltage activation process in Kv channels, offering broader implications for understanding channelopathies and designing targeted modulators.