Insight into tetrodotoxin blockade and resistance mechanisms of Na v 1.2 sodium channel by theoretical approaches.

Nav 1.2, a member of voltage-gated sodium channels (Nav s) that are responsible for the generation and propagation of action potentials along the cell membrane, and play a vital role in the process of information transmission within the nervous system and muscle contraction, is preferentially expressed in the central nervous system. As a potent and selective blocker of Nav s, tetrodotoxin (TTX) has been extensively studied in biological and chemical sciences, whereas the detailed mechanism by which it blocks nine Nav 1 channel subtypes remain elusive. Despite the high structural similarity, the TTX metabolite 4,9-anhydro-TTX is 161 times less effective toward the mammalian Nav 1.2, which puzzled us to ask a question why such a subtle structural variation results in the largely binding affinity difference. In the current work, an integrated computational strategy, including homology modeling, induced fit docking, explicit-solvent MD simulations, and free energy calculations, was employed to investigate the binding mechanism and conformational determinants of TTX analogs. Based on the computational results, the H-bond interactions between C4-OH and C9-OH of TTX and the outer ring carboxylates of the selectivity-filter residues, and the cation-π interaction between the primary amine of guanidinium of TTX and Phe385 determine the difference of their binding affinities. Moreover, the computationally simulations were carried out for the D384N and E945K mutants of hNav 1.2-TTX, and the rank of the predicted binding free energies is in accordance with the experimental data. These observations provide a valuable model to design potent and selective neurotoxins of Nav 1.2 and shed light on the blocking mechanism of TTX to sodium channels.