Computational Insights into the Dissociation Pathway of the Charybdotoxin–Shaker Complex: Voltage Dependency and Potassium Modulation

Abstract

The blockade of potassium channels by protein toxins is influenced by both voltage and permeant ions, yet the structural sequence of events leading to their unbinding is not resolved. Charybdotoxin (CTX) occludes the outer exit of the pore with Lys27 positioned in the S1 site, but the intermediate states that guide its dissociation remain unclear. Using long-timescale all-atom molecular dynamics, meta-eABF free-energy calculations, and residue-level contact analysis, we mapped the dissociation pathway of CTX from the Shaker channel. External electric fields triggered an early destabilization consistent with the voltage-dependent release of Lys27, giving rise to partially detached “wobbling” states. These intermediates involve coordinated axial and lateral motions of the toxin, supported by a small group of residues that maintain transient contacts with the channel vestibule. Under strong fields, K+ ions rapidly occupy S1 during these events, promoting Lys27 displacement and reducing the chance of rebinding S1. Contact patterns aligned with experimental Φ-values, distinguishing early- and late-breaking interactions. At the same time, the free-energy profile revealed a bound state and a wobbling intermediate separated by a modest reorganization barrier. Together, these results support a multistep dissociation mechanism shaped by voltage, K+ occupancy, and a dynamic network of residue contacts and provide an atomistic view of CTX unbinding from Shaker.