SUMMARY1. The experiments were done on single Ranvier nodes of Xenopus laevis (voltage clamp) and Rana esculenta (action potentials). Rate and size of the effect of tetrodotoxin were determined by the reversible reduction of either the sodium inward current (Xenopus) or of VN, the maximum rate of rise of the action potential (Rana).2. The results of tetrodotoxin block at equilibrium could be excellently fitted by assuming a one-to-one reaction between toxin molecules and sodium channels of the Xenopus membrane with an equilibrium dissociation constant K = 3-60 nm at room temperature. TA was not linearly related to the fraction of unblocked sodium channels and 10-9 nM tetrodotoxin was necessary on the average to reduce ,Ato 50 % in Rana motor fibres; in sensory fibres a lower concentration sufficed.3. Onset and offset of the tetrodotoxin effect on Xenopus nodes could be quantitatively interpreted as being determined by the rates of the tetrodotoxin channel reaction. Experiments with 3-1 and 15-5 nm tetrodotoxin at room temperature yielded an association rate constant, kj, of 2-94 x 106 m-1 sec-' and a dissociation rate constant, k2, of 1-42 x 10-2sec-1. In these experiments the equilibrium dissociation constant, K, was 3-31 nM. If determined solely from the onset in the two tetrodotoxin concentrations, ki = 3-25 x 106 M-1 sec-' and K = k2/kl = 4-08 nm was calculated.4. In Rana fibres the onset and offset of TA reduction by 15-5 and 31 nm tetrodotoxin was evaluated using the equilibrium effects of intermediary tetrodotoxin concentrations for calibration. The average results at room temperature were k, = 4 x 106 M-1 see', k2 = 1-4 x 10-2 sec-' and K = 3-4nM. 168 J. R. SCHWARZ, W. ULBRICHT AND H.-H. WAGNER 5. The very short latency with which IA started to decline when tetrodotoxin was suddenly applied proved that the toxin had ready access to the membrane.6. The temperature dependence of k1, k2 and K in the Xenopus experiments could be described by Arrhenius plots yielding activation energies, Ea, of 9-8, 20-5 and 7 0 kcal/mole, respectively, corresponding to Q10 values of 1-82, 3-42 and 1P53 (between 12 and 220 C). For k1, determined from onset alone, Ea = 13-7 kcal/mole (Q10 = 2.25) was obtained. Although in Rana the temperature dependence of the rate constants could not be determined directly, the Q10 for k2 must have been of the order of 3.7. The results suggest that the rate of the toxin action on the nodal membrane of Xenopus and Rana is limited by the tetrodotoxin-sodium site reaction.

1. Voltage clamp experiments were done on single myelinated nerve fibres of the frog, Rana esculenta. 2. 53 muM 4-aminopyridine (4-AP) reduced IK to about one-fifth if tested with infrequent (1/min) and short (10 ms) depolarizing pulses; the onset time constant under these circumstances was ca. 160 s (14-15 degrees C). After prolonged treatment the effect was virtually irreversible. 3. At equilibrium with 4-AP, increasing the frequency of short pulses removed part of the block, the block removal accelerating with increasing pulse duration and frequency. 4. In 53 muM 4-AP unblocking of K channels during long (0.8 s) depolarizing pulses proceeded with a time constant, taur, of ca. 0.2 s. Restoration of block at the resting potential proceeded with a much larger time constant, tau'r, of ca. 1 min. 5. The stationary fraction, rinfinity, of K channels conducting in 53 muM 4-AP was 0.66, 0.41, and 0.24 at V = 120, 50, and 0 mV, respectively. 6. In a series of experiments with [4-AP] varying between 13.3 and 848 muM, taur decreased from 0.25 to 0.10 s (V = 130 mV, ca. 17 degrees C) while rinfinity followed the empirical relation 1/rinfinity = 1 + ct + cv exp(-0.77 EF/RT) with E = V - 70 mV. ct and cv are dimensionless quantities that increase with [4-AP] and reflect the voltage-independent and voltage-dependent component, respectively, of block. 7. Block of K channels and partial removal are also observed with inward IK at raised [K+]O. Removal proceeds on depolarization even if IK is additionally but temporarily suppressed by tetraethylammonium. Hence neither direction nor amplitude of IK but only the pulse potential seems to determine the extent of block for a given [4-AP].

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