Cooperativity of tetrodotoxin action in the frog node of Ranvier

The steady state effects and rates of action of tetrodotoxin (TTX) on sodium current were studied in the voltage clamped frog node of Ranvier. Inactivation of the sodium current was separated into fast and slow phases. Both phases were assumed to correspond to two different currents (fast and slow) flowing through fast and slow channels (Benoit et al. 1985). The dose-response curve of the steady state effect of tetrodotoxin on the fast current was sigmoid. An analysis of this effect in double logarithmic coordinates gave a Hill coefficient of 1.74. The rates of tetrodotoxin action on the fast current were determined by the reversible reduction of the peak current recorded at a potential (+20 mV) at which the slow current was relatively small. After an initial delay, the onset of TTX effect followed an exponential function of time whose constant decreased with increasing tetrodotoxin concentrations. Expressed as the time corresponding to a reduction of 2% of the current, the delay (delta t2%) increased from about 100 ms with 300 nM-TTX to about 30 s with 1 nM-TTX. When tetrodotoxin was removed, the offset developed quasi-instantaneously and followed an exponential function of time whose constant was independent of the toxin concentration. Both steady state and rates of tetrodotoxin effects could be fitted well if one assumed that the block of one fast channel occurred after binding of two TTX molecules to two cooperative sites.