Tracking S4 Movements by Metal-Ion Bridges

Abstract

Electrical signalling in excitable cells depends on voltage-gated ion channels which open and close in response to changes in membrane potential. The voltage-gated ion channel consists of a central ion-conducting pore domain surrounded by four voltage-sensor domains (VSDs). The VSDs sense changes in membrane potential and confer this information to the pore domain. The fourth segment (S4) of each VSD carries several positively charged residues which gives the VSD its gating ability. S4 must traverse outwards through the membrane electric field in order for the channel to open. The open-state structures of both K and Na channels are known at atomic level through x-ray crystallography. In a previous investigation (Henrion et al., 2012, PNAS 109:8552-8557) we described four closed molecular configurations of a VSD based on 20 engineered metal-ion bridges, Rosetta modelling and molecular dynamics. A subset of these interactions was used to generate a detailed model of the intermediate conformations during VSD gating. Our results suggested that S4 slides >12 A along its axis during gating. Whether or not S4 continues to move after channel opening and during inactivation is not clear. Therefore, in the present study, we used the same technique, with double cysteine mutations and Cd ions, to explored molecular rearrangements in the activated state. Mutated Shaker K channels were expressed in Xenopus oocytes and studied by the two-electrode voltage-clamp technique. We found several new metal ion bridges suggesting possible S4 movements in the activated state.