1. The effect of various toxin fractions isolated by Watt et al. (1978) from the venom of the scorpion Centruroides sculpturatus Ewing on the Na currents of the node of Ranvier has been studied with the voltage clamp method. 2. The toxin fractions were applied externally. The most potent fractions were toxins III, IV and V which were effective in concentrations of 0.33-3.33 microgram/ml. The effect of toxins III and IV was quite different from that of toxin V. 3. In toxin III or IV - treated nodes a strong depolarizing pulse was followed by a transient shift of the negative resistance branch of the INa (E) curve to more negative potentials. The amount of shift varied between -10 and -60 mV. A 500 ms depolarizing pulse of small amplitude produced a slowly developing Na inward current which slowly decayed after the end of the pulse. Inactivation was incomplete, even with 500 ms pulses to 0 mV. 4. The transient shift of the INa (E) curve was not seen in nodes treated with toxin V. This toxin merely caused slow and incomplete Na inactivation. The effect of toxin IV was not suppressed by a four times higher concentration of toxin V, suggesting that the two toxins act on different receptors. 5. Toxin I acted like toxin IV but was about 10 times less potent. The effect of high concentrations of variants 1, 2, 3, 5, 6 resembled tha of toxin V. 6. All effects observed with toxin III or IV were also seen with the whole venom (cf. Cahalan 1975).

In voltage clamped nodes of Ranvier inactivation of the sodium permeability is slowed by toxin V from the scorpion Centruroides sculpturatus, by sea anemone toxin ATX II or by internally applied KIO3. The slow decay of the Na inward current is markedly accelerated if the test pulse is preceded by a depolarizing conditioning pulse followed by a 10-500 ms pause. This phenomenon was studied in detail, using conditioning pulses of varying amplitude and up to 15 s duration. In nodes treated with toxin V a 20 ms conditioning pulse to positive potentials was sufficient to produce a clear acceleration of the decay of the Na current and a reduction of the inward current remaining at the end of a 50 ms test pulse, i.e. a weakening of the toxin effect. In nodes treated with ATX II or internal KIO3 longer conditioning pulses were required. A similar effect of conditioning pulses on the decaying phase of the Na current was also observed in untreated fibres. To study the phenomenon quantitatively we fitted the decaying phase of the inward Na current with the equation INa = A exp(-t/tau 1) + B exp(-t/tau 2) + C. The effect of depolarizing conditioning pulses could be described as an increase of A, a decrease of B and C and a reduction of the time constants tau 1 and tau 2. I50/Ipeak, the normalised inward current remaining at the end of a 50 ms test pulse, decreased exponentially with increasing duration of the conditioning pulse to a steady-state value. The time constant tau and the steady-state value depended on the potential during the conditioning pulse. For nodes treated with toxin V, tau was 0.24 s at 0 mV and 12 degrees C and half inhibition occurred at -42 mV. The time constant tau was larger for nodes treated with ATX II or internal KIO3. At positive potentials, I50 was reduced to 20% of the control value in toxin V-treated nodes, but only to 70% in KIO3-treated nodes. Recovery from the effect of the conditioning pulse was studied by varying the pause between conditioning pulse and test pulse; recovery was 66-100% complete after 500 ms. The results are interpreted by assuming that a depolarizing conditioning pulse (a) accelerates inactivation of the sodium permeability and (b) causes dissociation of the toxin-receptor complex or transition into an inactive state. The latter effect occurs in toxin V-treated fibres but not in those treated with ATX II or KIO3.

The effect of several chemical reagents on the sodium current was studied in voltage-clamped single nerve fibers of the frog. The oxidants halazone and hypochlorous acid drastically inhibited inactivation. Their effect was similar to that of chloramine T (Wang, 1984a). The curve relating the steady-state inactivation parameter h infinity to the conditioning potential E became nonmonotonic after treatment with the oxidants, i.e., dh infinity/dE greater than 0 for E greater than -20 mV. By contrast, the oxidants periodate, iodate, and hydrogen peroxide (applied for the same time, but at higher concentrations) merely produced a parallel shift of the h infinity(E) curve to more negative values of membrane potential. Diethylpyrocarbonate, a reagent that preferentially modifies histidine groups, had one marked effect: a strong shift of the h infinity(E) curve to more negative values of membrane potential. Almost no effect was observed after application of the tyrosine-reactive reagent N-acetylimidazole. Similarly, the arginine-reactive reagent glyoxal had only minor effects on the Na permeability. The results suggest that methionine is not critically involved in the kinetics of Na current inactivation. Similarly, an essential tyrosine or arginine residue seems to be unavailable to chemical reagents from outside on the frog node of Ranvier. Deduced from the reactivities of (some of) the reagents used, modification of membrane lipids is a tentative explanation for the effects observed on inactivation kinetics.

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(1) Gating currents were recorded from frog nodes of Ranvier treated either with toxins III or IV from the venom of the scorpion Centruroides sculpturatus or with the alkaloid toxin aconitine. (2) Toxins III or IV from Centruroides sculpturatus (which drastically reduce the sodium permeability PNa and slightly shift its voltage dependence in the depolarizing direction) caused a small depolarizing shift of the relation between charge (Qon) and membrane potential (E) without affecting the maximum charge Qon max. (3) On nodes treated with toxins III or IV from Centruroides sculpturatus, a depolarizing conditioning pulse (which transiently shifts the descending branch of the INa(E) curve by up to 60 mV in the hyperpolarizing direction) shifted the midpoint potential (Emid) of the Qon(E) curve by -17 mV and slightly increased the slope of the curve; it also decreased Qon max markedly but had little effect on Qon measured with small depolarizing pulses. By contrast, massive treatment with aconitine (which irreversibly shifts sodium activation in the hyperpolarizing direction) irreversibly shifted the midpoint potential of the Qon(E) curve from -28.5 to -69 mV and significantly increased Qon and Qoff measured with small depolarizing pulses; concomitantly, the voltage dependence of the on time constant of the charge movement [tau on(E)] was shifted by -44 mV. (4) The sodium current INa was exponential both in nodes treated with toxins III or IV of Centruroides sculpturatus and subjected to a depolarizing conditioning pulse and in aconitine-treated nodes; in the latter, INa started after a delay of 30-40 microseconds. The time constant of the sodium current. tau on Na, was larger than the time constant of the charge movement, tau on Q; the ratio tau on Q/tau on Na was 0.61 and 0.73 in the experiments with Centruroides sculpturatus toxins and aconitine, respectively. (5) The off time constant of the sodium current (tau off Na) was slightly increased in nodes treated with Centruroides sculpturatus toxins and subjected to a depolarizing conditioning pulse, whereas it was markedly increased in aconitine-treated nodes. With the former treatment, the off time constant of the charge movement (tau off Q) was unaffected but with aconitine treatment it was considerably increased although it remained smaller than tau off Na. Consequently, the ratio tau off Q/tau off Na (which is greater than or equal to 1 in untreated nodes) became smaller than one, reaching values as low as 0.58 and 0.44 in the experiments with Centruroides sculpturatus toxins and aconitine, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)