Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis

The voltage-gated Naf channel cu-subunit consists of four homologous domain s arranged circumferentially to form the pore. Several neurotoxins, includi ng saxitoxin (STX), block the pore by binding to the outer vestibule of thi s permeation pathway, which is composed of four pore-forming loops (P-loops ), one from each domain. Neosaxitoxin (neoSTX) is a variant of STX that dif fers only by having an additional hydroxyl group at the N1 position of the 1,2,3 guanidinium (N1-OH). We used this structural variant in mutant cycle experiments to determine interactions of the N1-OH and its guanidinium with the outer vestibule. NeoSTX had a higher affinity for the adult rat skelet al muscle Na+ channel (mul or Scn4a) than for STX (DeltaG similar to 1.3 kc al/mol). Mutant cycle analysis identified groups that potentially interacte d with each other. The N1 toxin site interacted most strongly with mul Asp- 400 and Tyr-401. The interaction between the N1-OH of neoSTX and Tyr-401 wa s attractive (Delta DeltaG = -1.3 +/- 0.1 kcal/mol), probably with formatio n of a hydrogen bond. A second possible attractive interaction to Asp-1532 was identified. There was repulsion between Asp-400 and the N1-OH (Delta De ltaG = 1.4 +/- 0.1 kcal/mol), and kinetic analysis further suggested that t he N1-OH was interacting negatively with Asp-400 at the transition state. C hanges in pH altered the affinity of neoSTX, as would be expected if the N1 -OH site were partially deprotonated. These interactions offer an explanati on for most of the difference in blocking efficacy between neoSTX and STX a nd for the sensitivity of neoSTX to pH. Kinetic analysis suggested signific ant differences in coupling energies between the transition and the equilib rium, bound states. This is the first report to identify points of interact ion between a channel and a non-peptide toxin. This interaction pattern was consistent with previous proposals describing the interactions of STX with the outer vestibule (Lipkind, G. M., and H. A. Fozzard. 1994. Biophys. J. 66:1-13; Penzotti, J. L., G. Lipkind, H. A. Fozzard, and S. C. Dudley, Jr. 1998. Biophys. J. 75:2647-2657).