We will comment on Rilapladib distinct features of the PMF when we discuss the evolution of the distances between the contact pairs below. Using Eq. 1, we numerically integrate the PMF to find the binding constant and then determine the standard binding free energy using Eq. 2. The calculated value, {9:9 kcal/ mol, is in good agreement with the experimental value of {10:5 kcal/mol, which is determined from the most recent IC50 measurement where state-of-the-art equipment was employed. The level of agreement obtained for the standard binding free energy is similar to those obtained for potassium channel toxins, and Regorafenib HCl provides further support for the accuracy of the proposed model of the NaV1.4�C m -GIIIA complex. The binding mode of the NaV1.4�C m -GIIIA complex is dominated by charge interactions where the pairs are mostly at contact distances. Analysis of how the contact distances change during dissociation provides complementary information on the relative strength of the various interactions as well as helping to explain specific features of the PMF. For this purpose, we have calculated the average pair distances in each umbrella window and plotted them as a function of the window position. The toxin residues are seen to break up in the following order and at the approximate positions: K11 at 30 A ?, K16 at 34 A ?, R19 at 35 A ?, and finally R13 at 36 A ?. Because the weakest interactions will break up first and the strongest ones last, the persistence length of a pair during dissociation provides a good measure for its relative strength. This intuitive picture for the strength of the interactions is in good agreement with the mutation data in Table 2. For example, according to the set of data from, the affinity loss for mutations of K11, K16, R19, and R13 to Ala are, respectively, 10, 81, 199, and 642. In this work, we have constructed a homology model for the pore domain of the NaV1.4 channel by aligning the DEKA residues in the selectivity filter with the corresponding EEEE residues in NaVAb, whose crystal structure was determined recently.