Therefore, we have neglected these linker sequences in our current model of NaV1.4. While the S5-P1 linker faces the pore, it does not appear to be involved in binding of m -GIIIA, hence our results are unlikely to be affected by its absence. A 3D model of the channel is created using Modeller by threading the aligned NaV1.4 sequence for each domain on a corresponding domain of 3RVY. In order to refine the model and check its stability, we have performed MD simulations of the NaV1.4 model in a membrane environment. For this Iopamidol purpose, we have used the protocols established in previous MD simulations of ion Phenacetin channels. The NaV1.4 model is embedded in a lipid bilayer consisting of 153 POPE molecules in the x-y plane and solvated with a NaCl solution. Extra counter ions are included in the system to neutralize it where necessary. The system is then equilibrated in MD simulations in several stages. First the protein is fixed and the system is equilibrated with pressure coupling until the correct water and lipid densities are obtained. In order to get an adequate sampling of the side chain orientations, we use all ten NMR conformers of m -GIIIA in ensemble docking. Because there are no well-known binding motifs for NaV1 channel blockers��like the pore inserting Lys in potassium channel blockers��we have considered several possibilities for restraints in HADDOCK. To facilitate comparisons with the mutation data and simplify interpretation of the results, we use a single restraint in each docking study. The EEDD and DEKA ring of residues are the potential sites on the channel for using restraints. However, the mutation data indicates that the EEDD residues play a much more important role in binding of m -GIIIA than the DEKA residues. Therefore, only the EEDD ring is used as a restraint site in the following docking studies. The potential restraint sites on the toxin include the residues R1, K11, R13, K16, and R19, which are identified in mutagenesis experiments. Separate docking studies are performed for each of these residues and the EEDD ring as a restraint.