The three basic residues, R13, K16, and K11 are seen to weave around the EEDD ring, making multiple contacts with Pamidronate disodium pentahydrate residues in all four domains. We note that there is some redundancy here because two residues such as R13 and K16 can still cover all four domains consistent with the observation that m-GIIIA can also block the channel. The fact that two basic residues are sufficient to block the pore is also supported by several other m conotoxin blockers of NaV1 channels, which have only two basic residues available at the binding interface. Compared to potassium channels there are relatively fewer blockers of sodium channels, which is due to the larger pore size in the latter. A pore inserting Lys is sufficient to block a potassium channel whereas at least two basic residues are required to achieve the same in a sodium channel. The pore inserting Lys motif has been instrumental in functional studies of potassium channels using Ozagrel HCl toxins peptides as probes. This has simplified interpretation of experimental results as well as construction of computational models of channel�Ctoxin complexes. The situation in NaV1 channels is much more complicated due to many possible configurations for coupling of 2�C3 basic toxin residues with the EEDD residues in the pore. Also there are many high-affinity toxins that do not block NaV1 channels. These features have certainly made interpretation of mutation experiments a more difficult task and sometimes resulted in conflicting proposals for the binding modes. Construction of accurate complex models using homology models of NaV1 channels is expected to ameliorate this situation. Moreover such complex models will be very useful in designing analogues with enhanced affinity and selectivity properties, which may be required for development of toxin blockers of NaV1 channels as therapeutic agents. We have previously shown in over a dozen case studies involving potassium channel toxins that the binding free energy can be determined near chemical accuracy from PMF calculations.Thus calculation of the standard binding free energy of m GIIIA will provide a complementary test for the accuracy of the proposed NaV1.4�C m -GIIIA model. The PMF for the dissociation of m -GIIIA from NaV1.4 is constructed from umbrella sampling MD simulation as described in Methods.