The influence of pH on equilibrium effects of tetrodotoxin on myelinated nerve fibres of Rana esculenta.
1. The experiments were done on single nodes of Ranvier of Rana esculenta. The effects of tetrodotoxin and H ions were determined either by the reduction of the maximum rate of rise, VA, of action potentials evoked with threshold stimuli or in the voltage clamp by the decrease of the peak Na permeability, PNa. 2. With the tetrodotoxin sample used throughout the investigation the equilibrium dissociation constant, KT, of the toxin-receptor reaction at neutral pH was determined to be 2-8 nM. Between 1-55 and 15-5 nM tetrodotoxin the normalized value, A, of VA, was found to be related to the normalized toxin concentration cT = [TTX]/2-8 nM by the empirical equation log [(1-A)/A] = 1-22 log cT-0-573. 3. On increasing the pH (up to 8-8) the effect of tetrodotoxin diminished as revealed by an increase in A. The apparent reduction of cT (as calculated from A) suggests that the toxin is active only in its cationic forms. 4. Weakly acid tetrodotoxin solutions (7-3 less than pH less than or equal to 5-5) reduced A to a lesser degree than did neutral toxin solutions in spite of the inherent depressing effect of acid pH on A (A = 0-5 at about pH 5-5). In more acid toxin solutions A decreased again and at pH 4-6 it was about equal to the value in toxin-free solution. 5. When, after equilibrium in an acid toxin solution, the perfusate was suddenly changed to neutral Ringer solution A jumped to a higher value A' as measured 1 sec after the switch. Since the blocking effect of hydrogen ions subsided within a fraction of a second while the time constant of the toxin washout is of the order of 1 min, A' reflects the number of Na channels blocked by tetrodotoxin at acid pH. 6. In acid toxin-free solution the peak PNa as obtained in voltage clamp experiments was reduced by a voltage-dependent factor (cH + 1)-1 with CH = [H+]/KH(E) and KH(E) = 2-04 muM exp (0-34 EF/RT). Adding tetrodotoxin resulted in another reduction by a constant factor p'T. 7. Experiments employing various combinations of toxin concentration (3-1-93 nM) and pH values (7-3-5-2) confirm the decreased toxin effect at low pH. Moreover, p'T was smaller (the additional toxin effect larger) when the membrane had been kept depolarized and thus cH reduced during equilibration. This suggests that tetrodotoxin cations and H ions compete for the same blocking site. A quantitative fit, however, requires additional assumptions.
The influence of pH on equilibrium effects of tetrodotoxin on myelinated nerve fibres of Rana esculenta. 1. The experiments were done on single nodes of Ranvier of Rana esculenta. The effects of tetrodotoxin and H ions were determined either by the reduction of the maximum rate of rise, VA, of action potentials evoked with threshold stimuli or in the voltage clamp by the decrease of the peak Na permeability, PNa. 2. With the tetrodotoxin sample used throughout the investigation the equilibrium dissociation constant, KT, of the toxin-receptor reaction at neutral pH was determined to be 2-8 nM. Between 1-55 and 15-5 nM tetrodotoxin the normalized value, A, of VA, was found to be related to the normalized toxin concentration cT = [TTX]/2-8 nM by the empirical equation log [(1-A)/A] = 1-22 log cT-0-573. 3. On increasing the pH (up to 8-8) the effect of tetrodotoxin diminished as revealed by an increase in A. The apparent reduction of cT (as calculated from A) suggests that the toxin is active only in its cationic forms. 4. Weakly acid tetrodotoxin solutions (7-3 less than pH less than or equal to 5-5) reduced A to a lesser degree than did neutral toxin solutions in spite of the inherent depressing effect of acid pH on A (A = 0-5 at about pH 5-5). In more acid toxin solutions A decreased again and at pH 4-6 it was about equal to the value in toxin-free solution. 5. When, after equilibrium in an acid toxin solution, the perfusate was suddenly changed to neutral Ringer solution A jumped to a higher value A' as measured 1 sec after the switch. Since the blocking effect of hydrogen ions subsided within a fraction of a second while the time constant of the toxin washout is of the order of 1 min, A' reflects the number of Na channels blocked by tetrodotoxin at acid pH. 6. In acid toxin-free solution the peak PNa as obtained in voltage clamp experiments was reduced by a voltage-dependent factor (cH + 1)-1 with CH = [H+]/KH(E) and KH(E) = 2-04 muM exp (0-34 EF/RT). Adding tetrodotoxin resulted in another reduction by a constant factor p'T. 7. Experiments employing various combinations of toxin concentration (3-1-93 nM) and pH values (7-3-5-2) confirm the decreased toxin effect at low pH. Moreover, p'T was smaller (the additional toxin effect larger) when the membrane had been kept depolarized and thus cH reduced during equilibration. This suggests that tetrodotoxin cations and H ions compete for the same blocking site. A quantitative fit, however, requires additional assumptions.