Abstract

The large-conductance, calcium-activated potassium (BK) channel lacks the classic intracellular bundle-crossing gate observed in most other ion channels of the 6TM family. This observation, initially inferred from closed-pore accessibility experiments and recently corroborated by a cryo-EM structure of the non-conductive state, raises naturally a puzzling question: how can gating occur in absence of steric hindrance? To answer this question, we combined electrophysiology and molecular simulations and investigated the kinetics BK inhibition by two pore-blockers. The crux of our strategy was to leverage the state-dependent affinity of the binders to probe the physical properties of the pore. We thus combined kinetic modeling with a series of accurate free energy calculations to obtain a microscopic picture of the sequence of events taking place during the open-to-closed transition and giving rise to a nonconductive ..