Pharmacological Properties of Axonal Sodium Channels in the Cockroach Periplaneta Americana L.: II. Slowing of Sodium Current Turn-Off By Condylactis Toxin

It is established that tetrodotoxin (Pichon, 1969 a, b, 1974, 1976) and saxitoxin (Pelhate & Sattelle, 1974; Sattelle, Pelhate & Hue, 1979) specifically block the sodium channels in giant axons of the ventral nerve cord of the cockroach (Peri-planeta americana). No chemical probe specific for the sodium channel inactivation (closing) mechanism of insect axons is currently available. In the present study, extract from the tentacles of the marine coelenterate Condylactis gigantea, containing Condylactis toxin (CTX), a neurotoxic polypeptide (Shapiro, 1968 a), is tested on the axonal membrane of the cockroach (P. americana).

When applied to nerve bundles of a crayfish (Oronectes virilis), CTX produces repetitive firing of action potentials (Shapiro, 1968b). In addition, action potentials recorded from giant axons of the lobster (Homarus americanus) and from the stretch receptor cells of the crayfish (O. virilis) exhibit a prolonged falling phase at CTX concentrations of 0-2 mg/ml and above (Shapiro & Lilleheil, 1969). Voltage-clamp experiments on giant axons of another crayfish (Procambarus clarkii) indicate that these effects of CTX are due to a slowing of the turn-off of the transient sodium conductance (Narahashi, Moore & Shapiro, 1969). Subsequent analysis of the kinetics of sodium conductance changes in toxified crayfish (P. clarkii) axons under voltage-clamp have confirmed this view and show that the steady-state sodium inactivation curve is shifted in the direction of hyperpolarization ( Murayama et al. 1972). Another polypeptide toxin (ATX_II) has been purified by Béress et al. (1975) from a coelenterate (Anemonia sulcata). This toxin also prolongs action potential duration in giant axons of a crayfish (Astacus leptodactylus) by selectively slowing the sodium inactivation mechanism (Romey et al. 1976). ATX_n does not affect either the sodium activation or the steady-state potassium conductance (Romey et al. 1976). Prolonged exposure of crustacean axons to these anemone toxins results in conduction block with no more than a few millivolts depolarization of the axonal membrane (Shapiro & Lilleheil, 1969; Romey et al. 1976), Anemone toxins CTX and ATX_n therefore act on the mechanism by which the sodium channels of crustacean nerve membranes are closed.

The sensitivity of crustacean neurones to CTX and ATX_n contrasts sharply with the reported insensitivity to these toxins of molluscan neurones. For example, the giant axon of the squid is unaffected by CTX applied either externally (Narahashi et al. 1969) or internally (T. Narahashi, personal communication). In the same way, ATX_n is without effect on the giant axon of the cuttlefish (Sepia officinalis) when applied externally (Romey et al. 1976). It is of interest therefore to discover whether axons of arthropods other than crustaceans are also sensitive to CTX. In this study CTX is applied to giant axons of the cockroach (P. americana) which closely resemble other unmyelinated axons in both the passive permeability characteristics of their membranes and the mechanism of excitability (see Pichon, 1974). In particular, the application of voltage-clamp techniques to isolated cockroach giant axons has resulted in the demonstration of a transient, inward sodium current and a late, outward, potassium current during a depolarizing voltage-clamp pulse (Pichon, 1969 a, 1974, 1975). We have therefore examined the actions of CTX on isolated giant axons of the cockroach (P. americana) in order (a) to further assess the specificity of this toxin within the Arthropoda and (b) to attempt to extend the range of currently available specific chemical probes of the molecular mechanisms underlying excitation in insect axonal membranes.

Using the double oil-gap, single-fibre technique (Pichon & Boistel, 1967), voltageclamp and current-clamp experiments were performed on giant axons dissected from abdominal nerve cords of the cockroach (Periplaneta americana). The methods and recording techniques were as previously described (Pelhate, Hue & Chanelet, 1978; Sattelle et al. 1979). The ionic composition of normal saline was as described in the preceding paper (Sattelle et al. 1979). Voltage-clamp experiments were performed at 12 ±0·5 °C. For current-clamp experiments the temperature was in the range 20−22 °C. Condylactis toxin (CTX) was obtained from Sigma Chemical Co., Poole, Dorset U.K. (batch no. 126C-0277). The synthetic saxitoxin hydrochloride (STX) used in this study was the generous gift of Prof. Y. Kishi (Harvard University).

No effect was detected on either the resting potential or the action potential of an isolated giant axon when CTX was applied at concentrations of 0·1−0·3 ^mg/nd for 10 min. At 0·5 mg/ml and higher concentrations, CTX did not affect either the resting potential or the time-course and amplitude of the rising phase of the action potential, but the falling phase of the action potential was prolonged. Within 3−10 min of toxin application at a concentration of 0·5 mg/ml action potentials with an extended plateau in the falling phase were evoked (Fig. 1). The effects on the action potential of a 10 min exposure to CTX were reversed by washing the axon with normal saline for 30 min (Fig. 1).

The duration of the toxin-induced plateau in the falling phase of the action potential was prolonged by longer exposure to CTX (0·5 mg/ml) but was shortened when the stimulus frequency was increased. For example, in all axons tested increasing the stimulus frequency from 0·1 to 1·0 Hz attenuated the duration of the plateau potential (Fig. 2). When axons were stimulated at very low frequencies (0·01 Hz) plateau potentials of up to 0·8 s were recorded. Repetitive firing was not observed in CTX-treated axons when short (0·5 ms) depolarizing pulses were applied to the axonal membrane but 2−4 action potentials were sometimes observed during longer (50·6 ms) depolarizing pulses.

By the application of a series of depolarizing and hyperpolarizing pulses across the axon membrane it was possible to determine the current-voltage (I-V) relations for a single axon in normal saline and after a 5 min exposure to 0·5 mg/ml CTX (Fig. 3). A sharp break in the I-V curve was always noted in the presence of CTX. The CTX-induced changes in the I-V curve and the repetitive activity sometimes observed during long depolarizing pulses could be the result of a prolonged sodium current. To test the hypothesis that exposure of cockroach axons to CTX resulted in a prolonged sodium current, voltage-clamp experiments were performed.

The actions of CTX (0·5 mg/ml) on cockroach giant axons were tested under voltage-clamp conditions. Membrane currents were recorded in response to 4 ms step depolarizations to membrane potentials (E_m) of − 10 mV (first pulse) and + 40 mV (second pulse) from a holding potential (E_h) of − 60 mV. The twin pulse regime was repeated every two seconds. By this mean the actions of CTX on the total membrane currents and the late outward, potassium current were continuously monitored. As shown in Fig. 4 neither the amplitude nor the time to peak of the inward current was affected by CTX. The late current was however decreased by 19% when E_m= + 40 mV. Moreover when E_m= − 10 mV the late current, normally outward, became inwardly directed. The late inward current recorded when E_m= − 10 mV was maintained even when a longer depolarizing pulse (10 ms duration) was applied (Fig. 4 c). So although CTX weakly inhibited the late, potassium, outward current its major effect appeared to be a slowing of the sodium channel inactivation mechanism. To test this directly, the actions of CTX on the sodium current alone were examined.

Axons were first exposed to 10⁻⁴ M 4-aminopyridine for 5 min which suppressed almost all of the potassium current (cf. Pelhate & Pichon, 1974). The subsequent application of CTX (0·5 mg/ml) in the continued presence of 4-aminopyridine prolonged the sodium inward current without affecting the time required to reach peak inward current (Fig. 5).

It was also found that step depolarizations to E_m values more positive than the equilibrium potential for sodium ions elicited outward sodium currents which in the presence of CTX (0·5 mg/ml) remained outwardly directed throughout the duration of the depolarizing pulse (Figs 6, 7). Thus CTX was able to slow the closing of sodium channels of cockroach axons irrespective of the direction in which the sodium current was flowing through the membrane. After pretreatment of axons with CTX (0·5 mg/ml) resulting in prolonged inward sodium currents, the application at 10⁻⁸ M concentrations of the specific sodium channel inhibitor synthetic saxitoxin (Pelhate & Sattelle, 1978; Sattelle et al. 1979) in the continued presence of CTX, suppressed completely both the transient and the CTX-induced sodium currents. In two CTX-treated axons 10⁻⁷ M synthetic STX blocked respectively 80% and 90% of the peak inward sodium current. In one CTX-treated axon exposed to 2·10⁻⁸M synthetic STX, 80% of the peak inward sodium current was blocked. These effects of synthetic STX were not significantly different from those reported previously for synthetic STX in the absence of CTX (Sattelle et al. 1979). The sensitivity of the cockroach axon to synthetic STX was not, therefore, affected by pretreatment with CTX.

The present study has established that Condylactis toxin (CTX) induces a prolonged negative after-potential in action potentials recorded from giant axons of the cockroach Periplaneta americana. Action potentials with a distinct plateau potential (up to 0·8 s duration when the axon is stimulated at 0·01 Hz) can be recorded in the presence of CTX at 0·5 mg/ml and higher concentrations. In cockroach axons CTX acts primarily by slowing the turning-off of the sodium current, without affecting either the timecourse of sodium activation or the peak amplitude of the transient sodium current. An ∼ 20% decrease in the late outward potassium current of cockroach axons is also detected. The toxin therefore acts in a similar manner on the membranes of cockroach and crustacean axons (cf. Shapiro, 1968 b ; Shapiro & Lilleheil, 1969; Narahashi et al. 1969; Murayama et al. 1972). One difference has emerged between the actions of CTX on cockroach and other arthropod axons. The actions of CTX can be partly reversed in the case of cockroach axons whereas this is not the case for lobster (Homarus americanus) axons (Shapiro & Lilleheil, 1969). The sensitivity of arthropod axonal membranes to the anemone toxins CTX (Shapiro, 1968b; Shapiro & Lilleheil, 1969; Narahashi et al. 1969; Murayama et al. 1972) and ATX_II (Romey et al. 1976) and the insensitivity of cephalopod axons to the same toxins (Narahashi et al. 1969; Romey et al. 1976) remains to be explained. These contrasting results may reflect a restricted access of the toxins to the sodium channels of cephalopod axons. Another possible explanation is that there exist differences in the axonal sodium channel closing mechanisms between arthropods and molluscs.

Condylactis toxin provides a useful probe for the molecular mechanisms of sodium channel closing in arthropod axons. The MW of the toxin has been estimated to be in the range ∼ 10000−15000 daltons (Shapiro, 1968a). A recent investigation (Yost & O’Brien, 1978) has separated two components each of MW ∼ 5000 daltons both of which are toxic when injected into a terrestrial crustacean (Armadillidium vulgar e). The large size and the rapid action of CTX strongly suggest that it acts on the external surface of arthropod axonal membranes. Support for this view is provided by the observation that another anemone toxin (ATX_n) of MW ∼ 5000 isolated from Anemonia sulcata (Wunderer, Machleidt & Wachter, 1976; Abita et al. 1977) rapidly produces plateau potentials when iontophoretically applied to the external surface of a crayfish (Astacus leptodactylus) axon but fails to produce any effect when applied in the same manner to the cytoplasmic membrane surface (Romey et al. 1976).

The present study has established that exposure of the cockroach axonal membrane to CTX does not prevent access of saxitoxin (STX) to the sodium channel. Whether or not the two toxins act at different sites on the axonal sodium channel remains to be determined. The observed attenuation of the plateau potentials in response to an increased frequency of stimulation could be explained in several ways. For example, it may indicate that axonal activity inhibits either the binding of CTX to its receptor or the ability of the CTX-receptor complex to prolong the opening of the sodium channel. It is also possible that alterations in the ionic gradients across the membrane, slight changes in the resting potential, or even changes in the degree of inhibition of the potassium current could account for the observed effects of stimulation on the duration of plateau potentials.

A number of other chemicals have also been shown to prolong inactivation in cockroach axons. For example, Pichon (19696, 1976) has demonstrated that DDŸ induces complex permeability changes including a slowing of sodium inactivation. In addition it has recently been observed that allethrin prolongs sodium inactivation in cockroach axons (M. Pelhate, unpublished observations). CTX is nevertheless the most specific probe to date of the mechanism by which the sodium current of cockroach axons is turned off. Suitably radiolabelled it could provide a biochemical approach to the study of the inactivation mechanism of insect axonal membranes. The actions of CTX on cockroach axons described in this paper closely resemble the findings for all other arthropod axons tested. This correspondence points to a similar molecular mechanism underlying the closing of the axonal sodium channels in a variety of arthropod species.

The authors record their thanks to Profs D. Coullaud and J. Chanelet for the generous provision of laboratory facilities. The support of the Royal Society European Exchange Programme and a travel grant from ICI Ltd is gratefully acknowledged. The authors thank Professor T. Narahashi for permission to refer to his unpublished work.