Abstract
Voltage-gated ion channels enable electric signaling by responding to changes in membrane potential. This is controlled by four voltage-sensor domains (VSDs) in which the fourth transmembrane segment (S4) contains several positively charged residues that move in response to voltage changes. An open conformation is available from the Kv1.2/2.1 X-ray structure, and several recent simulations based on experimental constraints have lead to an emerging consensus model of the resting state. We have extended this approach by systematically exploring residue contacts that should occur during the VSD gating, and tested these with electrophysiology. By using metal-ion bridges that are weaker than disulphides it is possible to keep the channel working and quantify shifts in voltage dependence. We report a total of 20 new interactions, which more than double the number of experimental constraints available for VSDs, and classify them into one open and three successively more closed intermediates. A subset of constraints was used to build models of each conformation with Rosetta, and after subsequent simulation (without constraints) the models fulfill all constraints in each state. Further, under some conditions it appears to be possible to drive Shaker into an even deeper fourth closed state for which we also provide a model. Molecular simulations show that these intermediate states indeed correspond to meta-stable conformations. Starting from the first closed state and driving the S4 helix upwards in a simulation results in stable conformations within 3A RMSD of the experimental open state structure. These results provide insight both into the transition and intermediate states, and support our previous ideas of a 3(10)-helix region that moves in sequence in S4 in order to occupy the same position in space opposite F290 from the open through the three first closed states.
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