The voltage-gated Na+ channel is the central determinant of muscle excitability. There are seven human disorders associated with gene defects in the Na+ channel or its subunits including the Long QT Syndrome and idiopathic ventricular fibrillation. Drugs that act upon Na+ channels including anticonvulsants, local anesthetics, and antiarrhythmic agents. The aim of the proposed project is to test, refine, and expand a molecular model of the outer vestibule and selectivity filter of the Na+ channel as part of larger effort to understand the structural biology of this channel. Detailed structural information will be necessary to fully understand the function of the channel, to engage in rational drug design, and to consider the possibility of protein engineering in conjunction with gene therapy to ameliorate genetic diseases linked to the Na+ channel. The hypothesis being tested is that a detailed molecular model of a complementary binding surface can be constructed by determining sufficient ligand/substrate interaction points using several ligands of known structure. The experiments will evaluate, refine, and expand a previously described Na+ channel outer vestibule model by testing predictions about the points of interaction with high affinity ligands that bind in this area. The approach is general for ligand/receptor interactions, and an analogy for this approach would be a lock and key where the ligands are the keys and the Na+ channel is the lock. The structure of the lock is determined by looking at the shape of the keys. Specifically, points of interaction between channel amino acids and the ligands will be determined using mutant cycle analysis. The shape of the ligands and the points of interaction are used as constraints when refining computer-generated models of the binding surface. Multiple toxins will used to probe as much of the outer vestibule as possible, to serve as validation of results obtain with other ligands, and to develop sufficient interaction points to constrain adequately the model.