The simulation was allowed to continue for an additional 1 ns to allow the protein to equilibrate. For the final 1 ns, the average Cβ-Cβ distance had been reduced to 7.8 Å, in accordance with the expected distance of a cysteine metal bridge as found in the MDB. The backbone root-mean-square deviations (rmsd) between the initial model and the equilibrated model is 1.7 Å, as calculated in the transmembrane region, indicating that this CP-690550 constraint was satisfied with minimal rearrangement of the backbone atoms (see Figure 1A). A second model was constructed

according to the same method to reflect the observation of a separate Cd2+ bridge between residues R362C (S4) and I287C (S2) (Campos et al., 2007), corresponding to Kv1.2 residues R294C and I230C, respectively. The Cβ atoms of these residues were initially separated by a distance of 13.2 Å. Again, the metal bridge was formed by applying a harmonic restraint between the metal ion and the sulfur atom on the cysteine residues, therefore bringing the Cβ atoms to within 6.2 Å. The backbone rmsd between the initial and final conformation in the transmembrane region is 2.7 Å, indicating that the

interaction was formed with little movement of the protein backbone (see Figure 1B). Functional recordings of ionic currents have shown that magnesium (Mg2+) slows the kinetics of activation of the Shaker double mutants I287D in S2 and F324D in S3 (I230D and F267D in Kv1.2) but has little or no effect on deactivation (Lin et al., 2010). This behavior see more from is reminiscent of the voltage-sensing K+ channel Ether-a-go-go, which is known to contain a functional divalent cation-binding

site at those respective locations (Tang et al., 2000). Therefore, this site provides a separate constraint between S2 and S3 that must be satisfied in the resting state of the VSD. To examine the Mg2+ bridge between S2 and S3, we constructed a model based on the VSD of Kv1.2 in which the I230D and F267D mutations were introduced and an Mg2+ ion was inserted. In the initial model, the Cβ-Cβ distance between I230D-F267D is 5.2 Å, indicating that the Mg2+ bridge is readily satisfied without the need to alter the initial conformation of the VSD. Nevertheless, for methodological consistency, a 6 ns MD simulation was performed. The average Cβ-Cβ distance of the final 1 ns of simulation is 6.5 Å, and the backbone rmsd between the initial and final conformation is 1.2 Å (see Figure 1C). Functional recordings of ionic currents show that the resting state of the Shaker double mutant F290W-R362K (F233W-R294K in Kv1.2) is more energetically stable than the resting state of the single mutant F290W (Tao et al., 2010). Although the nature of the interaction was not identified, one possibility is that in the resting state, the double mutant enables an electrostatic interaction to occur between R1 and the acidic residue E2, which is one turn below the mutated phenylalanine.