In order to validate the NaV1.4 model, we have used the extensive functional data obtained from binding studies of m conotoxin GIIIA. An initial model for the NaV1.4 m-GIIIA complex is created using HADDOCK, which is refined in MD simulations. The binding mode obtained is in broad agreement with the available mutagenesis data and shows that the toxin blocks the pore through multiple interactions of the R13, K16 and K11 residues with the outer ring of EEDD residues in the channel vestibule. The standard binding free energy of m -GIIIA is determined from the PMF calculations and found to agree with the experimental value within chemical accuracy. Thus the proposed model of the NaV1.4�C m -GIIIA complex has been well validated. Because there is a high degree of homology among the NaV1 channels, the present NaV1.4 model can be used as a Testosterone propionate template in constructing homology models for the pore domain of other NaV1 channels. Our focus in this first study was the validation of the pore domain using the data on binding of m -GIIIA. For further studies of toxin binding to NaV1 channels one needs to include the selectivity filter and the S5-P1 linker in the model. For example, to investigate the ionic strength dependence of toxin binding, a validated model of the selectivity filter is required. This can be achieved by studying the permeation and selectivity properties of Na + ions, which we hope to tackle in a forthcoming paper. Our attempts to model the S5-P1 linker in the turret of the NaV1.4 channel have not been successful due to lack of good templates. Because binding of m -GIIIA does not appear to involve the S5-P1 linker residues, a satisfactory binding mode could still be obtained without modeling this region. However, the S5-P1 linker residues are involved in binding of some other m -conotoxins, and to understand the differences in their affinity and selectivity properties, it will be important to construct models of NaV1 channels including the full turret region. The available mutagenesis data could provide valuable Iproniazid phosphate guidance in this endeavor.