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Funnel web spider venom produces spontaneous action potentials in nerve
Life Sciences Vol . 20, pp . Printed in the II .S .A .
243-250, 1977 .
Pergamon Presa
FUNNEL WEB SPIDER VENOM PRODUCES SPONTANEOUS ACTION POTENTIALS IN NERVE Ian Spence, David J . Adams and Peter W . Gage School of Physiology and Pharmacology University of New South Wales Kensington, 2033, Australia (Received is final form November 10, 1976)
Venom from the lethal Australian spider, Atrax robustus, causes fasciculation of muscles in vivo and in so ate diaphragms in mice . Spontaneous en -p ate potentials were recorded in muscle fibres exposed to the venom and associated spontaneous electrical activity could also be recorded from the phrenic nerve . It was proposed that the venom produces muscle fasciculation by causing abnormal, spontaneous, repetitive firing of motor nerves . The mechanism of this action was investigated in aplysia neurones . The venom produced abnormal, spontaneous, repetitive inward currents in voltage clamped neurones and changed the current-voltage characteristics of the surface membrane . It is suggested that the basic mode of action of Funnel-web venom is to change the electrical field in nerve membrane . The Funnel-web spider, Atrax robustus, which is commonly found in New South Wales, Australia, is recogn se as a lethal species and several case histories have been reported describing signs of envenomation in affected patients (1,2) . A common early cause of death is respiratory insufficiency accompanying laryngeal spasm . This, and generalised muscle twitching which is often seen in affected patients, suggest that the venom causes abnormal contractions in skeletal muscle . In fact, injections of Funnel-web venom (FWV) into primates produce gross fasciculation which can be suppressed with muscle relaxants such as gallamine (2,3) . The origin of the abnormal contractions caused by crude Funnel-web venom has been examined in mouse phrenic nerve-diaphragm preparations, and in aplysia (Aplysia uliana) ganglia . Methods The initial series of experiments was carried out on isolated mouse phrenic nerve-diaphragm preparations maintained at 2730 ° C . In most of these experiments, preparations were continuously perfused with modified Krebs solution (pH 7 .4-7 .5) containing (mM) NaCI, 120 ; KC1, 3 .5 ; CaCI2, 2 .5 ; MgCl2, 1 ; NaHC03, 25 and glucose 11 . Solutions were continuously bubbled with 5~ CO2 1n 02 . Conventional electrophysiological techniques were used for intracellular recording from end-plate regions of muscle fibres . Microelectrodes were filled with 3M KC1 and had resistances of 25-30 M0 . Suction electrodes were used to stimulate and record action potentials from nerve fibres . 243
24 4
Funnel Web Venom on Nerve
Vol. 20, No . 2, 1977
In the second series of experiments, neurones R and R15 in the abdominal ganglion of aplysia (Aplysia ~ul~iana) were ~oltage-clamped using 3M KC1 electrodes (resistance 1-10 MS1) of r voltage recording and 2M potassium citrate electrodes (resistance 3-5 Mn) for passing current to control the membrane potential . The membrane current was recorded with an operational amplifier between the bath and ground . The physiological solution for the isolated aplysia ganglion contained (mM) NaCI, 494 ; KC1, 11 ; CaC12, 11 ; MgC12, 19 ; MgS04, 30 ; TRIS buffer, 10 ; (pH 7 .7) and was aerated throughout the experiment . Only crude venom from male spiders was used as it is more potent than venom from female spiders (1,2) . The venom, which was obtained from the Commonwealth Serum Laboratories, Melbourne, was added to mouse or aplysia physiological saline . The concentration used in all experiments was 10-5 g/ml and preparations were continuously perfused with solutions containing the venom . Results and Discussion Soon after exposure to FWV (within 2-8 minutes) isolated mouse diaphragms could be seen to fasciculate and occasionally, with higher concentrations of FWV, widespread contractures lasting several seconds occurred . Similar observations have been reported previously in isolated chicken biventer cervicis muscle (2) . The contractions could be suppressed with curare (d-tubocurarine, 13uM) so that it seemed unlikely that they were due to abnormal events in the muscle fibres themselves . Raising the extracellular magnesium concentration above 10 mM also abolished the contractions, as did tetrodotoxin (313 nM) . In such solutions in which fasciculation was suppressed it was possible to insert intracellular micro-electrodes into end-plate regions of muscle fibres .
Fig . 1 End-plat potentials recorded in a solution containing curare (1 .3uM) and FWV (10- g/ml) . In the upper trace, end-plate potentials were elicited in response to a single nerve stimulus at the time denoted by the stimulus artefact . The lower trace shows spontaneous end-plate potentials in an unstimulated preparation . The irregularity of the base-line was due to small spontaneous contractions throughout the muscle . Calibrations : vertical, 5 mV ; horizontal, 5 cosec .
Vol . 20, No . 2, 1977
Funnel Web Venon on Nerve
24 5
No change in the resting n~nbrane potential of muscle fibres exposed to FWV was detected . In 8 of 12 nerve-muscle preparations there was a wail elevation of the frequency of miniature end-plate potentials (MEPPa), by a factor of 2-B . This could have been due to depolarization of the nerve terminals or to another action on the secretion mechanism e .g . a depolarization-independent increase in calcium permeability of the nerve terminals . MEPPs had a normal time course and amplitude indicating that FWV did not affect acetylcholine receptors or the passive electrical properties of the muscle membrane . These results made it unlikely that FWV was producing fasciculation by causing a tonic depolarization of nerve terminals sufficient to elicit action potentials in the motor nerve . If it were, it might have been expected that muscle fibres would also be found to be depolarized . In solutions containing FI~iV (10 -5 g/ml) and curare (1 .3uM), end-plate potentials (EPPs) could be elicited by stimulating the phrenic nerve . These appeared essentially normal in time course and amplitude . However, after 2-60 min in the presence of FWV, a single stimulus to the nerve sometimes produced two or occasionally more EPPs as illustrated in Fig . 1 . This is not seen under normal conditions . In addition, spontaneous EPPs were recorded in some fibres when the phrenic nerve was not being stimulated (Fig . 1) . This extraordinary appearance of multiple or spontaneous EPP~ could be prevented by raising the magnesium concentration or by raising the calcium concentration in the extrace11u1ar solution .
C
Fig . 2 Extracellular recordings from mouse phrenic nerve showing that FNV causes spontaneous firing of nerve fibres that can be suppressed by raising calcium concentration . A . In control solution . B 8 C . After exposure t a solution containing FIiV (10-5 g/m1) . D . In a solution containing FWV (10-~ g/x~l) and raised calcium concentration (5 mM) . Calibrations : vertical, 100uV ; horizontal, 20 ursec .
24 6
Funnel Web Venom on Nerve
Vol . 20, No . 2, 1977
These observations pointed to the nerve as the site of action of FWV and indeed, extracellular recording from phrenic nerves exposed to FWV revealed spontaneously occurring brief currents (Fig . 2) which were presumed to be caused by action potentials in one or several axons . These could be abolished by tetrodotoxin (313 nM), by raising the calcium concentration to 5 mM or higher (Fig . 2) or by raising the magnesium concentration to more than 10 mM . It was concluded thât FWV causes muscle fasciculation by producing spontaneous action potentials in motor nerve fibres . This conclusion was supported by the results of experiments with aplysia neurones . When voltage-clamped neurones were exposed to FWV, abnormal, spontaneous, repetitive, inward currents were seen after ten minutes, as shown in Fig . 3A . The cell (R 5) was voltage-clamped at -45 mV, the normal resting membrane potential of his cell . Usually this cell is quiescent under such conditions and a potential of -45 mV can be maintained with a steady current (Fig . 3A, upper race) . When cells voltage-clamped at -45 mV were exposed to FWV (10 - g/ml), brief, repetitive inward currents were seen (Fig . 3A, lower trace) . The currents had the same time course and amplitude as sodium currents in these cells clamped at about -25 mV in normal aplysia solution (4,5) and could be blocked with tetrodotoxin (3 uM) .
A
B
Fi9 . 3 Effects of FWY (10 -5 g/ml) on voltage-clamped aplysia neurones " A : Currents recorded from a neurones volts e-clamped at -45 mV (R15) in control solution (upper trace) and in FWV solution lower trace) showing spontaneous inward currents in FWV . Calibrations : vertical, 1 uA ; horizontal, 1 sec . (18 ° C) . B : Currents recorded from a neurone in response to the depolarizing voltage step shown in the top trace (-42 mV to -18 mV) in control solution (middle trace) and in the presence of FWV (10 -5 g/ml) (bottom trace) . The peak sodium current recorded at -18 mV in control solution was 380 M and 470 nA in the presence of FWV . Calibrations : vertical, 100 mV and 2 uA ; horizontal, 100 msec .
vol . 20, No . 2, 1977
Fuanel Web Veaom an Nerve
24 7
It was concluded therefore, that the spontaneous, inward currents seen in the presence of FïiV were carried mainly by sodiNn ions . In one experiment, these spontaneous currents disappeared when the extracellular calcium concentration was raised from 11 mM (the normal concentration) to 55 mM . When a range of depolarizing steps was used, it became clear that, in the presence of FWV, less depolarization than normal was needed to elicit sodium currents . For example, clamping to a level of -18 mY produced an increase in peak sodium current in the presence of FWV (Fig . 3B, bottom trace) . The same phenomenon was seen with calcium and potassium currents as can be seen in Fig . 3B . In normal aplysia solution, depolarization to -18 mV produced inward sodium current but no inward calcium current (middle trace, Fig . 3B) . In contrast it was observed that depolarization to -18 mV in the presence of FWV (bottom trace, Fig . 3B) enerated a later, slower, inward ionic current, presumably calcium current ~5) . There was also a prominent outward potassium current in FWV, not seen nornially with voltage steps to -18 mV (5) (Fig . 3B) . This current could be blocked with tetraethylammonium ions (50 mM) . When peak sodium and potassium currents were plotted against clamp potential, it was seen that FWV shifted the current-voltage curves for sodium and potassium to more hyperpolarized potentials . A similar shift was seen in graphs of sodium and potassium conductance versus membrane potential . A plot of sodium conductance (gNa, calculated from the peak amplitude of sodium currents against clamp potential is shown in normal solution (filled circles and in the presence' of FWV (10 - 5 g/ml), (filled triangles) in Fig . 4 .
so
ao
so
(mV) F~ 4 The effect of FWY (10-5 g/ml) on peak sodium conductance (ordinate) as a function of clamp potential (abscissa) . In control solution (filled circles), the holding potential was -42 mV . In FWV solution, holding potentials of -42 mV (filled triangles) and -62 mV (open triangles) were used . In the former case, results were scaled to give the same peak sodium conductance maximum .
24 8
Fuanel Web Venom on Narve
Vol . 2G, No . 2, 1977
Measurements which were made with a holding potential of -42 mV revealed that the maximum sodium conductance was reduced by about 10~ in the presence of FWV . Therefore the points obtained for FWV (filled triangles) have been scaled to give the same gN~ maximum as in the control . Measurements shown as open triangles were obtained with a holding potential of -62 mV in the same neurone and are not scaled because the g maximum was the same as in control . Presumably, FWV caused inactivation ~ some sodium channels at a clamp potential of -42 mV, but not at -62 mV . However, the fact that there was no detectable change in the decay of sodium current suggests that there was no significant change in the rate of inactivation of sodium channels . The simplest explanation for the muscle fasciculation might have been that FWV caused depolarization of nerve membrane giving rise to repetitive firing of motor nerves . However, no depolarization of mouse diaphragm muscle or aplysia neurones was caused by the venom . This distinguishes FWV from batrachotoxin which produces depolarization of excitable membrane by increasing sodium permeability (6,7) . The effects of FhN, especially the shift to the left of current-voltage and conductance-voltage curves, are reminiscent of the effects of reducing extracellular calcium concentration (8) . The current voltage curves for sodium and calcium in aplysia neurones were shifted along the voltage axis by approximately 15 mV in the i~yperpolarizing direction in response to a five-fold decrease in the extracellular calcium concentration (9) . It is very unlikely that FWV chelates free calcium ions as evoked secretion of transmitter appeared normal at the mouse neuromuscular junction . Also, normal (4,5,9) inward calcium currents in response to depolarizing clamp pulses were recorded in aplysia neurones in the presence of FWV . On the other hand FWV may dislodge calcium from membrane binding sites or prevent calcium from screening fixed negative surface charge and so than e the membrane field seen by "voltage sensors" which gate ionic channels 10,11} . This might explain how the effects of FWV can be reversed by raising the calcium or magnesium concentration . However, the effect observed with high calcium could result from totally independent actions of calcium and FWV on ionic conductances . Another possibility is that FWV may bind to the membrane and modify the threshold for action potentials . Our working hypothesis is that FWV changes the electrical field within the membrane so that sodium channels are spontaneously activated although the traps-membrane potential is normal . The effects of FWV are similar in some respects to those of some scorpion venons . Both Buthus and Centruroides scorpion venoms cause repetitive firing of actinpotent a~nnerve fibres (12,13,14) . In frog myelinated nerve, the Centruroides venom causes a shift in current-voltage curves similar to that caused by FWV without depolarizing the surface membrane (13) . On the other hand a major action of Buthus venom on squid axons is to slow the rate of sodium inactivation . FWV does not seem to have the latter action as no slowing of the decay of sodium current was noted in its presence . It is interesting that the repetitive firing caused by Buthus venon (12) and by FWV is abolished by raising the extracellular calcium concentration (12) . Such toxins may provide tests for current models of membrane excitation and may give new insight into the normal events responsible for the initiation of action potentials in excitable cells . ACKNOWLEDGEMENTS The project was supported by a grant from the Clive and Vera Ramaciotti Foundations . We are grateful to Dr . S . Sutherland of Commonwealth Serum Laboratories for supplying the venom and to Mrs . C . Prescott for preparation of the manuscript .
249
Ftuiael Web Veaom on Nerve
Vol . 20, No . 2, 1977
REFERENCES
2. 3.
1.
S .K . SUTHERLAND, Medical Journal of Australia, 2 643-647 (1972) . S .K . SUTHERLAND, Anaesthesia and Intensive Care, 2, 316-328 (1974) . D . MORGANS, P .J . SPIRA, E .J . MYLECHARANE and W .E . BLOVER, Prop. Rust.
4. 5. 6.
D . GEDULDIG and R . 6RUENER, J. Physiot ., 211, 217-244 (1970) . D .J . ADAMS and P .W . GAGE, Science, 192, 783-785 (1976) . T . NARAHASHI, E . X, . ALBUQUERQUE and 7-bEGUCHI, J. gen. Physic! .,
7.
E .X . ALBUQUERQUE, I . SEYAMA and T . NARAFWSHI,
8.
B . FRAAKENHAUSER and A .L . HODGKIN,
9.
D .J . ADAMS and P .W . GAGE,
10 .
B . HILLE, A .M . WOODMILL and B .I . SHAPIRO,
11 .
S .G .A . McLAUGHLIN, G . SZABO and G . EISENMAN,
12 .
T . NARAHASHI, B .I . SHAPIRO, T . DE6UCHI, M . SCUKA and C .M . WANG, Am. a . PhysioZ ., 222, 850-857 (1972) . M .D . CAHALAN, J. Physik !London), 224, 511-534 (1975) . E . KOPPERHÖFER and H . SCHMIDT, PfZûgerâArch. ges . Physiot ., 303,
13 . 14.
Physi.ot . Phar~nrtcoZ . Soc ., 5, 234 (1974) . 54-70 (1971)
184, 308-314 (1973) . (1957) .
161P (1976) .
58,
J. Pharmaoot . Esp . Thsrap.,
J. Physic! : (London), 137, 218-244
Proo. Aunt . Physiot . PharmacoZ . Soc ., 7,
301-318 (1975) . 667-687 (1971) .
133-149, 150-161 (1968) .
Phit. Trans. Roy . Soc . B., 270, J . gen . Physiot ., 58,
243-250, 1977 .
Pergamon Presa
FUNNEL WEB SPIDER VENOM PRODUCES SPONTANEOUS ACTION POTENTIALS IN NERVE Ian Spence, David J . Adams and Peter W . Gage School of Physiology and Pharmacology University of New South Wales Kensington, 2033, Australia (Received is final form November 10, 1976)
Venom from the lethal Australian spider, Atrax robustus, causes fasciculation of muscles in vivo and in so ate diaphragms in mice . Spontaneous en -p ate potentials were recorded in muscle fibres exposed to the venom and associated spontaneous electrical activity could also be recorded from the phrenic nerve . It was proposed that the venom produces muscle fasciculation by causing abnormal, spontaneous, repetitive firing of motor nerves . The mechanism of this action was investigated in aplysia neurones . The venom produced abnormal, spontaneous, repetitive inward currents in voltage clamped neurones and changed the current-voltage characteristics of the surface membrane . It is suggested that the basic mode of action of Funnel-web venom is to change the electrical field in nerve membrane . The Funnel-web spider, Atrax robustus, which is commonly found in New South Wales, Australia, is recogn se as a lethal species and several case histories have been reported describing signs of envenomation in affected patients (1,2) . A common early cause of death is respiratory insufficiency accompanying laryngeal spasm . This, and generalised muscle twitching which is often seen in affected patients, suggest that the venom causes abnormal contractions in skeletal muscle . In fact, injections of Funnel-web venom (FWV) into primates produce gross fasciculation which can be suppressed with muscle relaxants such as gallamine (2,3) . The origin of the abnormal contractions caused by crude Funnel-web venom has been examined in mouse phrenic nerve-diaphragm preparations, and in aplysia (Aplysia uliana) ganglia . Methods The initial series of experiments was carried out on isolated mouse phrenic nerve-diaphragm preparations maintained at 2730 ° C . In most of these experiments, preparations were continuously perfused with modified Krebs solution (pH 7 .4-7 .5) containing (mM) NaCI, 120 ; KC1, 3 .5 ; CaCI2, 2 .5 ; MgCl2, 1 ; NaHC03, 25 and glucose 11 . Solutions were continuously bubbled with 5~ CO2 1n 02 . Conventional electrophysiological techniques were used for intracellular recording from end-plate regions of muscle fibres . Microelectrodes were filled with 3M KC1 and had resistances of 25-30 M0 . Suction electrodes were used to stimulate and record action potentials from nerve fibres . 243
24 4
Funnel Web Venom on Nerve
Vol. 20, No . 2, 1977
In the second series of experiments, neurones R and R15 in the abdominal ganglion of aplysia (Aplysia ~ul~iana) were ~oltage-clamped using 3M KC1 electrodes (resistance 1-10 MS1) of r voltage recording and 2M potassium citrate electrodes (resistance 3-5 Mn) for passing current to control the membrane potential . The membrane current was recorded with an operational amplifier between the bath and ground . The physiological solution for the isolated aplysia ganglion contained (mM) NaCI, 494 ; KC1, 11 ; CaC12, 11 ; MgC12, 19 ; MgS04, 30 ; TRIS buffer, 10 ; (pH 7 .7) and was aerated throughout the experiment . Only crude venom from male spiders was used as it is more potent than venom from female spiders (1,2) . The venom, which was obtained from the Commonwealth Serum Laboratories, Melbourne, was added to mouse or aplysia physiological saline . The concentration used in all experiments was 10-5 g/ml and preparations were continuously perfused with solutions containing the venom . Results and Discussion Soon after exposure to FWV (within 2-8 minutes) isolated mouse diaphragms could be seen to fasciculate and occasionally, with higher concentrations of FWV, widespread contractures lasting several seconds occurred . Similar observations have been reported previously in isolated chicken biventer cervicis muscle (2) . The contractions could be suppressed with curare (d-tubocurarine, 13uM) so that it seemed unlikely that they were due to abnormal events in the muscle fibres themselves . Raising the extracellular magnesium concentration above 10 mM also abolished the contractions, as did tetrodotoxin (313 nM) . In such solutions in which fasciculation was suppressed it was possible to insert intracellular micro-electrodes into end-plate regions of muscle fibres .
Fig . 1 End-plat potentials recorded in a solution containing curare (1 .3uM) and FWV (10- g/ml) . In the upper trace, end-plate potentials were elicited in response to a single nerve stimulus at the time denoted by the stimulus artefact . The lower trace shows spontaneous end-plate potentials in an unstimulated preparation . The irregularity of the base-line was due to small spontaneous contractions throughout the muscle . Calibrations : vertical, 5 mV ; horizontal, 5 cosec .
Vol . 20, No . 2, 1977
Funnel Web Venon on Nerve
24 5
No change in the resting n~nbrane potential of muscle fibres exposed to FWV was detected . In 8 of 12 nerve-muscle preparations there was a wail elevation of the frequency of miniature end-plate potentials (MEPPa), by a factor of 2-B . This could have been due to depolarization of the nerve terminals or to another action on the secretion mechanism e .g . a depolarization-independent increase in calcium permeability of the nerve terminals . MEPPs had a normal time course and amplitude indicating that FWV did not affect acetylcholine receptors or the passive electrical properties of the muscle membrane . These results made it unlikely that FWV was producing fasciculation by causing a tonic depolarization of nerve terminals sufficient to elicit action potentials in the motor nerve . If it were, it might have been expected that muscle fibres would also be found to be depolarized . In solutions containing FI~iV (10 -5 g/ml) and curare (1 .3uM), end-plate potentials (EPPs) could be elicited by stimulating the phrenic nerve . These appeared essentially normal in time course and amplitude . However, after 2-60 min in the presence of FWV, a single stimulus to the nerve sometimes produced two or occasionally more EPPs as illustrated in Fig . 1 . This is not seen under normal conditions . In addition, spontaneous EPPs were recorded in some fibres when the phrenic nerve was not being stimulated (Fig . 1) . This extraordinary appearance of multiple or spontaneous EPP~ could be prevented by raising the magnesium concentration or by raising the calcium concentration in the extrace11u1ar solution .
C
Fig . 2 Extracellular recordings from mouse phrenic nerve showing that FNV causes spontaneous firing of nerve fibres that can be suppressed by raising calcium concentration . A . In control solution . B 8 C . After exposure t a solution containing FIiV (10-5 g/m1) . D . In a solution containing FWV (10-~ g/x~l) and raised calcium concentration (5 mM) . Calibrations : vertical, 100uV ; horizontal, 20 ursec .
24 6
Funnel Web Venom on Nerve
Vol . 20, No . 2, 1977
These observations pointed to the nerve as the site of action of FWV and indeed, extracellular recording from phrenic nerves exposed to FWV revealed spontaneously occurring brief currents (Fig . 2) which were presumed to be caused by action potentials in one or several axons . These could be abolished by tetrodotoxin (313 nM), by raising the calcium concentration to 5 mM or higher (Fig . 2) or by raising the magnesium concentration to more than 10 mM . It was concluded thât FWV causes muscle fasciculation by producing spontaneous action potentials in motor nerve fibres . This conclusion was supported by the results of experiments with aplysia neurones . When voltage-clamped neurones were exposed to FWV, abnormal, spontaneous, repetitive, inward currents were seen after ten minutes, as shown in Fig . 3A . The cell (R 5) was voltage-clamped at -45 mV, the normal resting membrane potential of his cell . Usually this cell is quiescent under such conditions and a potential of -45 mV can be maintained with a steady current (Fig . 3A, upper race) . When cells voltage-clamped at -45 mV were exposed to FWV (10 - g/ml), brief, repetitive inward currents were seen (Fig . 3A, lower trace) . The currents had the same time course and amplitude as sodium currents in these cells clamped at about -25 mV in normal aplysia solution (4,5) and could be blocked with tetrodotoxin (3 uM) .
A
B
Fi9 . 3 Effects of FWY (10 -5 g/ml) on voltage-clamped aplysia neurones " A : Currents recorded from a neurones volts e-clamped at -45 mV (R15) in control solution (upper trace) and in FWV solution lower trace) showing spontaneous inward currents in FWV . Calibrations : vertical, 1 uA ; horizontal, 1 sec . (18 ° C) . B : Currents recorded from a neurone in response to the depolarizing voltage step shown in the top trace (-42 mV to -18 mV) in control solution (middle trace) and in the presence of FWV (10 -5 g/ml) (bottom trace) . The peak sodium current recorded at -18 mV in control solution was 380 M and 470 nA in the presence of FWV . Calibrations : vertical, 100 mV and 2 uA ; horizontal, 100 msec .
vol . 20, No . 2, 1977
Fuanel Web Veaom an Nerve
24 7
It was concluded therefore, that the spontaneous, inward currents seen in the presence of FïiV were carried mainly by sodiNn ions . In one experiment, these spontaneous currents disappeared when the extracellular calcium concentration was raised from 11 mM (the normal concentration) to 55 mM . When a range of depolarizing steps was used, it became clear that, in the presence of FWV, less depolarization than normal was needed to elicit sodium currents . For example, clamping to a level of -18 mY produced an increase in peak sodium current in the presence of FWV (Fig . 3B, bottom trace) . The same phenomenon was seen with calcium and potassium currents as can be seen in Fig . 3B . In normal aplysia solution, depolarization to -18 mV produced inward sodium current but no inward calcium current (middle trace, Fig . 3B) . In contrast it was observed that depolarization to -18 mV in the presence of FWV (bottom trace, Fig . 3B) enerated a later, slower, inward ionic current, presumably calcium current ~5) . There was also a prominent outward potassium current in FWV, not seen nornially with voltage steps to -18 mV (5) (Fig . 3B) . This current could be blocked with tetraethylammonium ions (50 mM) . When peak sodium and potassium currents were plotted against clamp potential, it was seen that FWV shifted the current-voltage curves for sodium and potassium to more hyperpolarized potentials . A similar shift was seen in graphs of sodium and potassium conductance versus membrane potential . A plot of sodium conductance (gNa, calculated from the peak amplitude of sodium currents against clamp potential is shown in normal solution (filled circles and in the presence' of FWV (10 - 5 g/ml), (filled triangles) in Fig . 4 .
so
ao
so
(mV) F~ 4 The effect of FWY (10-5 g/ml) on peak sodium conductance (ordinate) as a function of clamp potential (abscissa) . In control solution (filled circles), the holding potential was -42 mV . In FWV solution, holding potentials of -42 mV (filled triangles) and -62 mV (open triangles) were used . In the former case, results were scaled to give the same peak sodium conductance maximum .
24 8
Fuanel Web Venom on Narve
Vol . 2G, No . 2, 1977
Measurements which were made with a holding potential of -42 mV revealed that the maximum sodium conductance was reduced by about 10~ in the presence of FWV . Therefore the points obtained for FWV (filled triangles) have been scaled to give the same gN~ maximum as in the control . Measurements shown as open triangles were obtained with a holding potential of -62 mV in the same neurone and are not scaled because the g maximum was the same as in control . Presumably, FWV caused inactivation ~ some sodium channels at a clamp potential of -42 mV, but not at -62 mV . However, the fact that there was no detectable change in the decay of sodium current suggests that there was no significant change in the rate of inactivation of sodium channels . The simplest explanation for the muscle fasciculation might have been that FWV caused depolarization of nerve membrane giving rise to repetitive firing of motor nerves . However, no depolarization of mouse diaphragm muscle or aplysia neurones was caused by the venom . This distinguishes FWV from batrachotoxin which produces depolarization of excitable membrane by increasing sodium permeability (6,7) . The effects of FhN, especially the shift to the left of current-voltage and conductance-voltage curves, are reminiscent of the effects of reducing extracellular calcium concentration (8) . The current voltage curves for sodium and calcium in aplysia neurones were shifted along the voltage axis by approximately 15 mV in the i~yperpolarizing direction in response to a five-fold decrease in the extracellular calcium concentration (9) . It is very unlikely that FWV chelates free calcium ions as evoked secretion of transmitter appeared normal at the mouse neuromuscular junction . Also, normal (4,5,9) inward calcium currents in response to depolarizing clamp pulses were recorded in aplysia neurones in the presence of FWV . On the other hand FWV may dislodge calcium from membrane binding sites or prevent calcium from screening fixed negative surface charge and so than e the membrane field seen by "voltage sensors" which gate ionic channels 10,11} . This might explain how the effects of FWV can be reversed by raising the calcium or magnesium concentration . However, the effect observed with high calcium could result from totally independent actions of calcium and FWV on ionic conductances . Another possibility is that FWV may bind to the membrane and modify the threshold for action potentials . Our working hypothesis is that FWV changes the electrical field within the membrane so that sodium channels are spontaneously activated although the traps-membrane potential is normal . The effects of FWV are similar in some respects to those of some scorpion venons . Both Buthus and Centruroides scorpion venoms cause repetitive firing of actinpotent a~nnerve fibres (12,13,14) . In frog myelinated nerve, the Centruroides venom causes a shift in current-voltage curves similar to that caused by FWV without depolarizing the surface membrane (13) . On the other hand a major action of Buthus venom on squid axons is to slow the rate of sodium inactivation . FWV does not seem to have the latter action as no slowing of the decay of sodium current was noted in its presence . It is interesting that the repetitive firing caused by Buthus venon (12) and by FWV is abolished by raising the extracellular calcium concentration (12) . Such toxins may provide tests for current models of membrane excitation and may give new insight into the normal events responsible for the initiation of action potentials in excitable cells . ACKNOWLEDGEMENTS The project was supported by a grant from the Clive and Vera Ramaciotti Foundations . We are grateful to Dr . S . Sutherland of Commonwealth Serum Laboratories for supplying the venom and to Mrs . C . Prescott for preparation of the manuscript .
249
Ftuiael Web Veaom on Nerve
Vol . 20, No . 2, 1977
REFERENCES
2. 3.
1.
S .K . SUTHERLAND, Medical Journal of Australia, 2 643-647 (1972) . S .K . SUTHERLAND, Anaesthesia and Intensive Care, 2, 316-328 (1974) . D . MORGANS, P .J . SPIRA, E .J . MYLECHARANE and W .E . BLOVER, Prop. Rust.
4. 5. 6.
D . GEDULDIG and R . 6RUENER, J. Physiot ., 211, 217-244 (1970) . D .J . ADAMS and P .W . GAGE, Science, 192, 783-785 (1976) . T . NARAHASHI, E . X, . ALBUQUERQUE and 7-bEGUCHI, J. gen. Physic! .,
7.
E .X . ALBUQUERQUE, I . SEYAMA and T . NARAFWSHI,
8.
B . FRAAKENHAUSER and A .L . HODGKIN,
9.
D .J . ADAMS and P .W . GAGE,
10 .
B . HILLE, A .M . WOODMILL and B .I . SHAPIRO,
11 .
S .G .A . McLAUGHLIN, G . SZABO and G . EISENMAN,
12 .
T . NARAHASHI, B .I . SHAPIRO, T . DE6UCHI, M . SCUKA and C .M . WANG, Am. a . PhysioZ ., 222, 850-857 (1972) . M .D . CAHALAN, J. Physik !London), 224, 511-534 (1975) . E . KOPPERHÖFER and H . SCHMIDT, PfZûgerâArch. ges . Physiot ., 303,
13 . 14.
Physi.ot . Phar~nrtcoZ . Soc ., 5, 234 (1974) . 54-70 (1971)
184, 308-314 (1973) . (1957) .
161P (1976) .
58,
J. Pharmaoot . Esp . Thsrap.,
J. Physic! : (London), 137, 218-244
Proo. Aunt . Physiot . PharmacoZ . Soc ., 7,
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