This analysis converges toward an average computationally derived consensus model that is consistent with a wide range of available experimental data. The overall conformation that satisfies all the available constraints appears to be defined within 3 Å root-mean-square (rms) for the backbone atoms, which indirectly reflects the semiquantitative “resolution” of the available knowledge of the resting state, as interpreted

via MD simulations. These results help to better circumscribe www.selleckchem.com/products/nu7441.html the current views of voltage sensing and highlight the emerging consensus regarding the resting-state conformation of a VSD in Kv channels. Our aim is to examine the structural implications from four experimental residue-residue interactions known to occur in the resting-state conformation. Our starting point is the model of the Kv1.2 channel proposed by Pathak et al. (2007), which was subsequently 3-Methyladenine purchase refined by Khalili-Araghi

et al. (2010) using all-atom MD simulations of a membrane environment in explicit solvent. Previous calculations of the sensing charge using this model resulted in a value of 12–13 elementary charges per tetramer (Khalili-Araghi et al., 2010), consistent with experimental estimates observed in Shaker channels (Aggarwal and MacKinnon, 1996, Seoh et al., 1996 and Schoppa et al., 1992). For the sake of simplicity, only a single VSD embedded in a solvated bilayer is considered. Four all-atom models of the VSD were constructed and simulated. In each model, specific site-directed mutations were introduced, and harmonic restraints were applied to steer the model toward a configuration in which the interaction is realized. The results are shown in Figure 1, and those interactions are discussed below. The overall deviations of the models are shown in Figure S1 available online. Functional recordings of the gating current in Shaker have identified pairs of cysteine residues that

are amenable to metal bridge formation via a cadmium (Cd2+) ion (Campos et al., 2007). The cysteine-cysteine Tolmetin Cd2+ bridge involves residues R362C (S4) and I241C (S1), corresponding to Kv1.2 residues R294C in S4 and I177C in S1. To examine the Cd2+ bridge between S1 and S4, we constructed a model by introducing the mutations R294C and I177C in the resting-state model of the Kv1.2 VSD using the PsfGen module of the program VMD. The cysteine residues were introduced in the deprotonated form (carrying a charge of −1), and a Cd2+ ion was inserted. In the initial model, the Cβ atoms of these residues are 12.2 Å apart. However, the Metalloprotein Database and Browser (MDB) (Castagnetto et al., 2002) shows that cysteine pairs bridged by Cd2+ ions exhibit a Cβ-Cβ distance of roughly 5–7 Å. Therefore, the Cβ-Cβ distance was initially too large for a metal bridge to be formed.