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

The voltage-gated Na⁺ channel α-subunit consists of four homologous domains arranged circumferentially to form the pore. Several neurotoxins, including saxitoxin (STX), block the pore by binding to the outer vestibule of this permeation pathway, which is composed of four pore-forming loops (P-loops), one from each domain. Neosaxitoxin (neoSTX) is a variant of STX that differs 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 skeletal muscle Na⁺ channel (μl or Scn4a) than for STX (ΔG ∼ 1.3 kcal/mol). Mutant cycle analysis identified groups that potentially interacted with each other. The N1 toxin site interacted most strongly with μl Asp-400 and Tyr-401. The interaction between the N1-OH of neoSTX and Tyr-401 was attractive (ΔΔG = -1.3 ± 0.1 kcal/mol), probably with formation of a hydrogen bond. A second possible attractive interaction to Asp-1532 was identified. There was repulsion between Asp-400 and the N1-OH (ΔΔG = 1.4 ± 0.1 kcal/mol), and kinetic analysis further suggested that the N1-OH was interacting negatively with Asp-400 at the transition state. Changes in pH altered the affinity of neoSTX, as would be expected if the N1-OH site were partially deprotonated. These interactions offer an explanation for most of the difference in blocking efficacy between neoSTX and STX and for the sensitivity of neoSTX to pH. Kinetic analysis suggested significant differences in coupling energies between the transition and the equilibrium, bound states. This is the first report to identify points of interaction 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.