Nature Utilization Of The Slow Inactivation Mechanism In Voltage Gated K+ Channels

Voltage gated K+ channels gate in response to changes in the electrical membrane potential by the coupling of a voltage sensing module with a K+-selective pore. During prolonged depolarizations, these channels enter a non-conductive slow-inactivated state that involves constriction of the channel pore. Recent studies suggest that the slow inactivated state is triggered by the penetration of water molecules into well-defined peripheral cavities surrounding the selectivity filter. Here we describe Cs1, a cone-snail toxin that blocks the drosophila shaker channel using a novel mode of action that shares common features with its inherent slow inactivation. Using double-mutant cycle analysis complemented by unconstrained docking and refinement by molecular-dynamics we obtained a docked toxin model, in which the toxin is bound off the pore axis. Analysis of the MD data suggested that Cs1 does not block the access of water molecules to the pore, but greatly modifies the access of water to the peripheral cavities. In concert with recent studies, we demonstrate that water permeation into these cavities is governed by a network of hydrogen-bonds formed at the extracellular face of the channel. The toxin specifically target these hydrogen bonds in three different channel subunits, by either eliminating or stabilizing them. The resulting imbalanced movement of water molecules around the selectivity filter triggers a series of molecular events culminating in an asymmetric collapse of the selectivity filter. A series of electrophysiological experiments utilizing D2O and various toxin/channel mutants that support the proposed mechanism will be described.