Equilibrium and Kinetic Properties of the Interaction between Tetrodotoxin and the Excitable Membrane of the Squid Giant Axon

Squid giant axons were treated with tetrodotoxin (TTX) in concentrations ranging from 1 nM to 25 nM and the resulting decrease in sodium current was followed in time using the voltage clamp technique. The removal of TTX from the bathing solution produced only partial recovery of the sodium current. This suggests that the over-all interaction is more complex than just a reversible reaction. By correcting for the partial irreversibility of the decrease in sodium current, a dissociation constant of 3.31 x 10^-9 M was calculated for the reaction between TTX and the reactive site of the membrane. The data obtained fit a dose-response curve modified to incorporate the correction for partial irreversibility when calculated for a one-to-one stoichiometry. The fit disagreed with that calculated for a reaction between two molecules of TTX with a single membrane-reactive site, but neither supported nor disproved the possibility of a complex formed by two reactive sites with one molecule of TTX. Values of the rate constants for the formation and dissociation of the TTX-membrane complex, k ₁ and k ₂, respectively, were obtained from the kinetic data. The values are: k ₁ = 0.202 x 10⁸ M ^-1, and k ₂ = 0.116 min^-1. The magnitude of the dissociation constant derived from these values is 5.74 x 10^-9 M, which has the same order of magnitude as that obtained from equilibrium measurements. Arrhenius plots of the rate constants gave values for the thermodynamic quantities of activation.