The rate of action of tetrodotoxin on myelinated nerve fibres of Xenopus laevis and Rana esculenta

Abstract

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.

Drug blockade of open end‐plate channels.

“…This similarity suggests that the structural variation between these molecules does not importantly influence f, which is probably mainly governed by diffusion of the barbiturate molecule to a rather inaccessible intrachannel site (see Discussion). It is also interesting to note that these values forf are very similar to that determined by Schwarz, Ulbricht & Wagner (1973) for tetrodotoxin binding to frog node. The great difference in the affinity of tetrodotoxin and barbiturates stems entirely from the much lower rate of dissociation in the former case.…”

Tetrodotoxin binding to normal depolarized frog muscle and the conductance of a single sodium channel.

“…2B shows that the action potential of frog muscle is no less sensitive to tetrodotoxin than that of frog myelinated nerve (Schwarz, Ulbricht & Wagner, 1973 of normal resting potentials. The failure rate was already 13 % out of twenty-four fibres at 10 nm in the experiment of Fig.…”

“…The drug tetrodotoxin blocks sodium currents and action potentials in nerve and muscle at extremely low concentrations (Hille, 1968;Cuervo & Adelman, 1970;Schwarz, Ulbricht & Wagner, 1973) and has no other known effects on excitable membranes. It is therefore reasonable to assume that at sufficiently low concentrations, the drug binds only to sodium channels, and this suggests that one can determine the number of sodium channels in excitable membranes by examining their capacity W. ALMERS AND S. R. LEVINSON for binding tetrodotoxin (Moore, Narahashi & Shaw, 1967;Keynes, Ritchie & Rojas, 1971).…”

Failure of Chronic Denervation to Alter the Sensitivity of the Rat Diaphragm to Local Anaesthetics or Quinidine

“…The use of the maximum rate of rise of the action potential as an index of the blocking effect of drugs on the sodium channels is not altogether satisfactory, since there will undoubtedly be a non-linear relationship between the fraction of channels blocked and fraction by which Vmax is decreased. This has been shown, for example, by Schwarz, Ulbricht & Wagner (1973) on the node of Ranvier of frog myelinated nerve fibres where a 50% block of the maximum rate of rise of the action potential requires a considerably higher concentration of TTX (10.9 nM) than is needed to decrease by 50% the maximum depolarization-induced increase in sodium conductance, GNa (3.6 nM).…”