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ACTIONS OF THREE STRUCTURALLY DISTINCT SEA ANEMONE TOXINS ON CRUSTACEAN AND INSECT SODIUM CHANNELS ViNcENT L . SALGADo' *

and

WiLLIAM R . ~

'Rohm and Haas Company, 727 Norristown Road, Spring House, PA 19477, U .S.A.; and 2Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Box 100267, Health Science Center, Gainesville, FL 32610, U .S.A . (Received 3 February 1992; accepted 18 June 1992)

and W. R. KiEm. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 13651381, 1992 .-The membrane actions of three recently isolated polypeptide neurotoxins from the sea anemones Stichodactyla helianthus (toxin Shl), Condylactis gigantea (toxin Cgln and Cafactis parasitica (toxin Cpl) were investigated on action potentials and voltage-clamp membrane currents of the giant axon of the crayfish Procambarus clarkii. The first two toxins were also tested on the cockroach (Periplaneta americana) giant axon . All three toxins were particularly lethal to crustaceans, moderately toxic to an insect (cockroach), and essentially non-toxic to a mammal (mouse). Shl and CgH were 50- to 100-fold more potent on crayfish than on cockroach axons; this difference in activity was correlated with the relative reversibility of their effects on these arthropod axons. The crustacean selectivity of these toxins is therefore due largely to their greater affinity for crustacean sodium channels . All three toxins prolonged crayfish giant axon action potentials by selectively slowing Na channel inactivation without greatly affecting activation . Before toxin treatment, inactivation was nearly exponential, with a time constant less than 1 cosec. After treatment, the inactivation time course could be described as the sum of two exponentially decaying components, plus a large steady-state component. The major component possessed the slower (10-20 cosec) time constant . The steady-state component increased with depolarization, causing the sodium channel steady-state inactivation curve to reach a minimum between - 60 and - 20 mV and then increase at more positive potentials . All three toxins shitted the peak sodium current-voltage relation to the left . This voltage shift was greater at 20°C than at 10°C . Maintained membrane depolarization during toxin wash-in delayed the appearance of modified Na channels. Also, prolonged depolarization of toxin-treated axons converted modified sodium channels back to normal ones. The toxins did not affect potassium and leakage currents . Our results indicate that the three crustaceanactive sea anemone toxins share a common electrophysiological action on arthropod sodium channels, at least at the macroscopic level. V. L . SALGADO

" Presentittle] ress: DowFlanco Discovery Research, P.O. Box 708, Greenlleld, IN 46140, U .S.A . t Author to whom correspondence should be addressed . 1365

1366

V. L. SALGADO and W. R. KEM INTRODUCTION

(5000 mol. wt) sea anemone polypeptide neurotoxins vary tremendously in their toxicity, particularly against mammals (SCHwEiTz et al., 1981, 1985 ; KEM, 1988a, b; KEM et al., 1989). Anemonia and Anthopleura toxins isolated from anemones belonging to the family Actiniidae were initially selected for investigation due to their relatively high mammalian toxicity (SHmATA et al., 1974). More recently, other anemone toxins possessing high crustacean but low mammalian toxicity have been isolated and sequenced (CAmELLO et al., 1989; KEM et al., 1989). Although some Condylactis (family Actinüdae) crustacean-active toxins have much sequence similarity with the above-mentioned mammalian toxins, the sequences of the Stichodactyla and Calliactis toxins are quite distinct from the toxins isolated from the sea anemone family Actiniidae. In addition to sequence differences, it has been found that stichodactylid toxins are immunochentically distinct from actiniid toxins (ScHw= et al., 1985 ; KEM et al., 1989). ScHwErTz et al. (1985) also reported that the stichodactylid Radianthus (now named Heteractis) toxins were unable to displace specific binding of radioiodinated toxins AsII* and AsV to rat brain synaptosomes, even though they did displace scorpion a-toxin binding. The actiniid (type 1) sea anemone toxins displayed a significantly higher affinity for cardiac and denervated skeletal muscle tetrodotoxin (TM resistant Na channels, compared with neuronal and skeletal muscle Na channels having high TTX-sensitivity (CATTERALL and COPPERSMITH, 1981 ; RENAUD et al., 1986). However, this difference in sensitivity was not apparent when the type 2 Heteractis toxins were tested on these mammalian preparations (ScHwErTz et al., 1985 ; RENAUD et al., 1986). Together, these results suggested that there may be two or more sea anemone polypeptide binding sites on sodium channels. In view of these reported differences in structural, immunochemical, and pharmacological properties between the type 1 actinüd and type 2 stichodactylid toxins, we compared the electrophysiological actions of two recently isolated toxins upon crayfish and cockroach giant axons. CgII is the most abundant type 1 toxin isolated from the actiniid Condylactis gigantea, whereas Shl is the most abundant type 2 toxin present in Stichodactyla helianthus. While the crayfish axon component of this study was still in progress we received a third anemone toxin, calitoxin or CpI (CARMLLO et al., 1989). Since the sequence of this toxin differed considerably from those of the type 1 and 2 toxins, we decided to investigate its effects upon crayfish axon ion currents . THE LARGE

METHODS Microekctrode experiments Crayfish (Procambarur clwkit) nerve cords were isolated between the brain and subesophageal ganglion, deaheathed, and perfused with normal van Harreveld's saline containing, in mM : 205 NaCl, 5.4 KC1, 10 CaC1 2, 2.6 MgC1 2 and 3 HEPES buffer, adjusted to pH 7.5. The medial giant axons were impaled with glass microelectrodes possessing a resistance of 7 to 10 MEL Methods for cockroach (Per(laneta amerieana) giant axons were similar to those for crayfish axons: we used unidentified giant axons in the desheathed abdominal nerve cord, perfused with a bicarbonate-buffered cockroach saline containing, in mM : 210 NaCI, 3.1 KCI, 5.4 *Nomenclame-AsIl, Anemonia sukata toxin II; AsV, Anemonia sulcata toxin V; CgII, Condylactis gigantea toxin II ; ShI, Stichodactyla hefianthus toxin I; Cpl, Cafactis parasitica toxin I; HmI, Heteractis maerodactyhts toxin I.

Sea Anemone Toxins

1367

CaCl2, 0.5 NaHZPO4 and 2.1 NaHC0 3, adjusted to pH 7.2 . All microelectrode experiments were done at 22-23°C, with continuous perfusion. Voltage-clamp experiments Crayfish giant axons were voltage clamped with an axial wire method modified from Swum (1974), using solutions based on LuND and NARAHAm (1981). The nerve cord was dissected from ganglion 2 (subesophageal ganglion) to ganglion 10 (numbered according to SHRAOIIt, 1974), and placed in an ice-cooled Plexiglas acrylic chamber in potassium-free external solution (below) for desheathing and cleaning. During cleaning, the cord was held taut between two clips with # 8 silk suture in such a way that it could be rotated on its axis. The medial giant axon on one side of the cord was cleaned of adhering fibers, especially between ganglia 6 and 9, which would be in the current-measuring region. The other side of the cord was left intact for support, with tufts of connecting fibers in the region of the ganglia. Although it was often possible to obtain goad preparations from isolated axons, the success rate was improved with the partial cleaning described. The aircumesophageal connectives (between ganglia 1 and 2) are more easily dissected than the portion of the nerve cord between ganglia 2 and 10, and the giant axon is usually of the greatest and most uniform diameter in that region, making it suitable for use in the sucrose gap voltage clamp method (SALOADo et al., 1986). However, the axon was invariably leaky in the region of ganglion 2, making it impossible to obtain a long enough stretch of good axon in this region for the axial wire method. The cleaned nerve cord was transferred to a Plexiglas® acrylic voltage clamp chamber, aligned parallel to (and within 100 pen of) three platinized platinum plate electrodes consisting of a 2-mm wide central current-measuring electrode flanked by two grounded 5-mm guards (refer to SHRAc3=l, 1974, for diagram) . A perfusion cannula pulled from glass capillary tubing was inserted at one end of the cleaned axon, between ganglia 9 and 10, and the axon was tied to it with # 10 silk suture. The other end of the nerve cord was pulled around two posts made from 250 um silver wires glued to the bottom of the chamber, and the suture tied to that end was fixed to the outside wall of the chamber with Tackiwax® soft wax (Central Scientific Co ., Franklin Park, IL, U.S.A .) after the region of the axon between ganglia 7 and 8 was positioned near the central platinum plate. A slit was made in the axon where it rounded the bend around the first silver post, and the internal perfusion was begun with a pressure head of 3-4 cm. Often, momentary application of higher pressure was needed to get perfusion started. The internal electrode was of the 'piggy-back' design, consisting of a 25-Mm Pt-10% Ir wire glued to a capillary that had been pulled to about 401mm from larger tubing (WPI no-fil 1.0 mm). A stiff copper wire soldered to the tip of a soldering iron provided localized heat to attach the wire to the capillary with hot-melt craft glue. The capillary was long enough so that the tip could reach the center of the current-measuring electrode, and the wire protruded 6mm past it to reach the end of the further guard electrode. The wire was platinized for the last 12 mm (6 mm along the capillary and 6mm beyond it), except for a small region near the capillary tip where the two were glued. Finally, a 10-Min Pt-10% It wire was platinized and inserted into the capillary from the very tip to the shank, to reduce the high frequency impedance of the electrode. This internal wire was essential for a rapid voltage-clamp system . The capillary and an external reference electrode that was positioned near the axon in the current-measuring region were filled with 1 M KCl and connected to a differential amplifier with Ag/AgCI pellet electrodes . Most voltage-clamp experiments utilized a two pulse protocol ; the first pulse lasted 30 msec and was used not only to obtain the transient (Na) I-V relation, but also to serve as a conditioning pulse for the subsequent 10 mace test pulse to -10 mV, used to determine h. The two pulses were separated by a 150 psec recovery interval when the membrane was damped to the holding potential. All experiments were carried out using potassium-free external saline (concentrations, in mM : 210 NaCl, 10 CaCl2, 2 HEFES, pH 7.5), and internal saline (mM; 15 NaCl, 50 CsF, 170 Cs glutamate, 5 HEFES, pH 7.35) . However, to avoid depolarizing the axon, the internal perfusion was begun with an internal solution containing K+ in place of Cs+, until the voltage was damped.

TOXbZ3

ShI was purified as previously described (KEm et al., 1989) from the Caribbean sun anemone, Stichodartyla helianthus. CgII, the major neurotoxin variant of the Caribbean pink-tipped sea anemone, Condylactis gigantea, was similarly purified except that an additional anion exchange chromatography step was used to separate the isotoxins; the purification and characterization of CgIl and the other Condylactis neurotoxins will be reported separately (KEt et al.. in preparation) . Calitoxin (CpI), isolated from the acontiate European anemone Calliactis parasitica, was graciously provided by Dr L. CAttmLLO; its purification and properties have been reported (C Anrm .0 et al., 1989). Freeze-dried samples of the toxins were dissolved in van Harreveld's or cockroach saline; aliquots of these solutions were kept frozen at -20°C prior to use. Cockroach toxicity was assessed by injection of aqueous toxin solutions into the thorax of adult male P. tamerlcma. A two-fold dose interval was used and at least 10 animals were injected at each dose, which never exceeded a volume of 10 pl. Lethality was assessed for all three species 24 hr after injection. A moving average

1368

V. L. SALGADO and W. R. KEM TABLE 1 . CouPARATwE Toxlcarv oP THE Toxm To cRABs, CDC[RiOACHBS AND laM. COCpLOACH DATA VMRE DETERIQI M IN THE PREmNT sruDY, As DEscRmw IN MsrmoDs. TIm 95% coNPmENcE INTERVALS ARE SHOWN IN PARENTE EMS LDSo(1Iglkg) Toxin

Crab

Cockroach

Mouse

C91I SM CpI

0.20 0.3 0 20.Ot

780 (630-980) 3600 (2400-5300) 14,000 (10,000-19,000)

> 50,0000 > 15,000' > 15,000

*KEài (198ßa); tCARn3LLO et al. (1989) . method was used to estimate LD,e values (THompsm and WEU, 1952). Since only a small sample of CpI was available, only three 20 g Swiss-Webster male mice were mtrapentoneally injected with 0.3 mg of this toxin; none of the mice were paralyzed or died over a 24-hr period.

RESULTS

Phyletic differences in sensitivity to sea anemone neurotoxins

The relative toxicities (LD50) of the three sea anemone polypeptides for crab, cockroach, and mouse are summarized in Table 1 . All three toxins were 100-10,000 times more toxic to crabs than to cockroaches. The symptoms of intoxication displayed by cockroaches and crabs were qualitatively similar: initial tremors of the legs were followed by convulsions, spastic limb contractions, flaccid paralysis, and finally death. All three toxins were nontoxic to mice, when i.p. injected at very high doses. Microelectrode studies

Toxin ShI prolonged the action potential in crayfish axons in a concentration- and timedependent manner. At 4 nM (near the threshold concentration), the first observed change was the appearance of a negative afterpotential which often produced one or more extra spikes (Fig. IA). This negative afterpotential grew larger with time, and a plateau appeared at -30 to -40 mV. While this plateau was generally less than 10 msec in duration, the negative afterpotential lasted much longer. At 20 nM, ShI produced a similar early time course of changes, but after longer exposure to the toxin the plateau lasted as long as 250 msec, and was accompanied by depolarization and subsequent block . The action potential repolarization phase often of the action potential (Fig. 1B-D) proceeded through more than one plateau level (Fig. 1C). The effects of Shl on crayfish axons were irreversible on the time scale (about 2 hr) of these experiments. In cockroach axons, Shl also produced a distinct plateau phase but this developed in a different manner, as a negative afterpotential that gradually increased in amplitude (Fig. 2A), rather than as a plateau that began at a certain amplitude and increased in duration . Shl strongly depolarized the cockroach axon and induced repetitive firing (Fig. 2A). Reversibility of Shl action upon the insect axon is illustrated in Fig. 2B. Condylactis toxin (4 nM) also prolonged the falling phase of the crayfish giant axon action potential, producing a plateau at -30 to -40 mV, which gradually increased in duration to a maximum of about 40 cosec. However, there was minimal maintained depolarization at this concentration (Fig. 3A). At 20 nM, Cgü produced more complex

Sea Anemone Toxins

1369

20

E

-20

e-40 -60 -80

. 1 ,0 Time (msec)

A.

C.

0 .0

0.1

0 .2 0 .3 Time (sec)

0

B.

0.4

2 4 Time (msec)

D-

6

0.0 0.2 Time (sec)

Fyo. 1 . PRoLonoATtoN o -nn? Acnox PoTal mAL uv CRAYFISH bMuL GIANT Moons By Shl . (A) 4 nM Shl prolonged the action potential and blocked it at 70 min. The dotted trace was recorded before treatment, and subsequent traces were recorded at 8-min intervals, beginning 30 min after treatment . Traces shown were recorded at 8-min intervals. (B-D) 20 nM Shl . B is an expanded version of C, which shows prolonged action potentials, a slight tendency for repetitive firing, and transitions between three plateau potentials. (D) After prolonged exposure to 20 nM ShI the action potential shortened and was eventually blocked . Records in B-D were taken at 2min intervals . The apparent early temination of some of the traces was due to the fact that the sweep duration had to be increased during the experiment as the plateau duration increased . Temperature, 23°C .

action potentials, with oscillations between two plateau levels lasting as long as 130 msec, followed by a small negative afterpotential, during which there was a chance of a second spike (Fig. 3B and C). Similar effects of a crude Cg toxin sample upon lobster giant axons were first reported by SHAPIRo and LILLSFISII. (1969). Eventually, conduction of the action potential was blocked when the resting potential was depolarized by about 15 mV. As with Shl, the effects of CgII on crayfish axons could not be reversed by prolonged washing with toxin-free saline (data not shown) .

1370

V. L. SALGADO and W. R. KEM

E E >

2

4 6 Time (msec)

8

no . 2 . PROLONGATION OF THE ACTION POTEN77AL IN A COCKROACH GIANT AXON BY 200 nM Shl . (A) The first trace is the control, and the second, which differs only slightly, was taken after 60 min exposure to 100 nM Shi. The remaining traces were taken at 2-min intervals after addition of 200 nM SM, and show the gradual rise of the plateau, with repetitive firing occurring at certain times . (B) Washing with toxin-free saline led to almost complete reversal of the effects within 76 min . Traces shown in panel B began 10 min after the last trace in panel A ; the first four were taken every 4 min, and subsequent ones were taken every 20 min . Temperature, 23°C .

The time courses of ShI and CgII action upon resting and action potentials of the crayfish giant axon are shown in Fig. 4. Although the first noticeable effect was action potential prolongation, this is not clear in Fig. 4 because of the scale. Both toxins also gradually depolarized the axons, and when the depolarization was sufficient, conduction was blocked. Comparison of Figs 1 and 4 indicates that the action potential duration was prolonged more by Shl than it was by CgII. Table 2 summarizes the microelectrode data for these toxins on both arthropod axon preparations . The results can be summarized by stating that both ShI and CgII prolong the action potential, induce a negative afterpotential, and depolarize the membrane. However, ShI had a greater tendency to induce a negative afterpotential and resting membrane depolarization . This toxin also had a greater tendency to produce repetitive firing . Both ShI and CgII were 50- to 100-fold less active on cockroach than on crayfish axons, and their effects were readily reversible on the cockroach axon .

Sea Anemone Toxins

137 1

204 nM Cgll -20-'

_-20-

36 min

E E-4060

-80

-80

A,

0

20

40

Time (msec)

-1 0 0 -L,L_, 0 B.

2

. 4

'

Time (msec)

6

FIG . 3 . PROLONGATION OF THE ACTION PO7EN17AL IN CRAYFISH àG:DIAL GIANr AXONS BY CgIl . (A) 4 nM Cgll was applied at time 0, and tram shown were taken at 0, 12, 24, 27, 30, 33 and 36 min. (B) 20 nM CgII was applied at time 0, and records were taken every 2 min . (C) Continuation of B, showing shortening of the plateau, secondary action potentials, oscillations between two plateau levels, membrane depolarization, and eventually, conduction block . Temperature, 23°C.

Voltage-clamp studies on crayfish giant axons: general observations

Figure 5A shows Na currents obtained before and after treatment with 100 nM ShI. At -60 mV, where the control Na current was small, there was a large Na current in the presence of toxin. The Na current activated with a normal time course at all potentials, but inactivated much more slowly . The peak current-voltage relations for this experiment are shown in Fig. 5B . The activation midpoint was shifted to the left by 14.6 mV in the presence of Shl, reflecting the larger currents at negative potentials. When the control Na current was maximally activated, between 0 and +4 mV, the peak was larger than it was in the presence of toxin. This was due to rundown of the Na current during toxin treatment in this particular axon; peak current was usually increased early during toxin treatment. Inactivation of the Na current, though greatly slowed, reached a maximum

1372

V.

L. SALGADO and

W. R.

KEM

40

250

20

E

D

200 «0 a c 1

0

io c

-20 c. m -40

150 R 0

0

E 0

100q

-60 -80

-100

0

A.

40

120

80

time (min)

-0- resting potential -A- peak potential -0- AP duration

20

D

0

0-

c 1 w

2-20 0 Q. 0

0

-40

c Co -60

3 N 0

E

É -80 -100

B.

0

40

120

80

time (min)

corm 20 nM ShI (A) AND CgII (B) AcwN ON cRAYP15H GIANT AXON RPSrnaa POTENTIAL, ACTION POTENTIAL PEAK, AND ACTION POTENTIAL DURATION. The missing point in panel A is due to conduction failure . Temperature, 20°C .

Fro . 4 . Tnm

TABLE 2. CompmtmoN OF Tim ePPECrs OF ShI AND Cg1I ON cRAYPIsH AND oocKRoAcH TOXIN AxoNs, As DLmmsmvED BY ormAcELLULAR wcRacaLBCmovE Rscownvo . TmE MINIMUM EI+ECTIVE CONCENTRATION (MW POR EACH TOXIN EPPECT WAS THE LOWEST coNcENTRATiON TESTED THAT PRODUCE THE EPPECT WmaN 40 min. THE TEST ooNcEKTRATIONS wEaE 2, 4, 20, 40, 200 AND 400 nM . MEC (nM) Species Crayfish Crayfish Cockroach Cockroach

Toxin

Plateau

Depolarize

CgII ShI CgII ShI

4 4 400

20 4 400

200

200

Block 20

4 > 400 > 400

Reversible no no yes yes

Sea Anemone Toxins

A. -60 mV

0 .01

0 N

E

-0 .5-

á -1 .0

_

-40 mV 1 mA/cm2 5 msec o

(20 MV

137 3

,,

-2 .0

B.

-80

-40 0 Vm (MV)

40

FIG. 5 . EFFECT of 100 nM 3h1 ON A VOLTAGE CLAWED CRAYFISH CHANT AXON. Sodium currents at different test pulse potentials. (B) Peak Na current-voltage relations for the same axon . (C) Steady-state Na channel inactivation . The graph shows normalized peak current for test pulses to -10 mV following 30 mom conditioning pulses to the indicated potentials, with a 150 ps recovery interval between the conditioning and test pulses . Experiment V0118 ; Temperature, 20°C. (A)

between -60 and -20 mV (Fig. 5A); at more positive potentials it became less and less complete. There were clearly two components of inactivation in the toxin-treated axon. The slower component became smaller with depolarization beyond -20 mV, while the size of the fast component did not seem to vary with clamping potential. Toxin effects on the steady-state level of sodium channel inactivation are reflected in the h. curves of Fig. 5C, which were measured with a standard 2-pulse inactivation protocol with a 30 msec conditioning pulse. In the presence of toxin, h. reached a minimum of about 0.2 between -60 and -20 mV, then increased again at more positive potentials . The effects of CgII upon the Na current (Fig. 6) were very similar to those of ShI. They also resembled the effects previously observed by MuRAYAMA et al. (1972) with a crude Condylactis toxin extract, but differed in that a hyperpolarizing shift in the voltage dependence of activation was observed in our experiment. In the present study, which utilized a pure Cg toxin sample, a decrease in potassium current was never observed. For ShI and CgII a number of similar voltage clamp experiments were carried out at both 10 and 20°C, and a single experiment with CpI was performed at 10°C. The CpI data

1374

V. L. SALGADO and W. R. KEM

FIG . 6. EFFECT OF 20 nM Cg II ON A VOLTAGE CLAMPED CRAYFISH GIANT AXON . Sodium currents before and after treatment with 20 nM CgIL (H) Peak Na current-voltage relations for the same axon. (C) Steady-state Na channel inactivation . Experiment V0131; Temperature, 20°C . (A)

TABLE 3. EFFECTS OF THE THREE SEA ANEMONE TOXINS ON THE VOLTAGE-DEPENDENCE OF THE PEAK SODIUM CONDUCTANCE. A Vv , THE SIFT IN THE MIDPOINT OF THE NEGATIVE SLOPE R>KiION OF THE PEAK CURRENT-VOLTAGE RELATION, WAS MANUALLY DETERI[DIED FROM THE PLOTTED IN RELATION

Toxin

Experiment

ShI

V9605 V9606 V9607A V0118

CgII

V9606A V0202A V0131 V9607

CpI

V0202

Temperature °(C)

Concentration (nM)

10 10 10 20 20 10 20 20 10

40 100 . 100 100 100 200

40 20 40

A Vv

(mV) -4 5 -6 -15 -12 -8 -14 -il 3

Sea Anemone Toxins

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TABLE 4. EFFECTS OF THE THREE sEA ANEMONE TOxw ON 9oDHJM CHANNEL INACTIVATION. MmE co1 awANTs (T) AND RELATIVE AmpLrruDEs (A) WERE oBTAeaED BY DOUBLE ExpomENTUL Fris of THE ENACTTVATION MO; COURSE. WHERE DATA ARE MIMMO, DOUBLE EXPONENTIAL Fris WERE NOT ADEQUATE . THE suBsCR m ARE 9 FOR sLOw, f FOR FAST AND 99 FOR STEADY-sTA7E . AMPLITUDES wERE EXTRAPOLATED TO ZERO MŒ

Toxin/ Experiment/ Temperature Shl V0118 20°C CgII V0131-1 20°C CPI V9607 101C

Vm (mV)

A,

(mew)

AI

(mew)

A,

0 -10 10 20 10 0 20 30 0 -10 10 20 30 40

0 .49 0 .72 0.45 0.38 0.52 0.58 0.40 0 .34 0.64 0.69 0 .57 0 .41 0.38 0.35

12.25 12.40 17 .00 20.30 15.06 12.91 15 .18 17.52 10.35 20.23 15.17 10.67 10.10 13 .13

0.28 0.24 0.30 0.32 0.26 0.24 0.29 0.33 0.11 0.17 0.15 0.13 0.13 0.16

0 .76 0 .94 0 .79 0 .73 0 .71 0 .87 0 .67 0 .59 1 .23 1 .54 1 .17 1 .06 1 .00 1 .28

0.22 0.03 0.25 0.30 0.22 0.18 0.32 0.33 0.25 0.13 0.28 0.46 0.50 0.49

T,

TI

shown in Fig. 7 are typical for all three toxins at the lower temperature. At 10°C activation was only weakly shifted to the left, as reflected by the currents in Fig. 7 at - 50 and -30 mV and the current-voltage relations in Fig. 7B. Inactivation in the presence of CpI had a greatly slowed time course, but its voltage dependence was very similar to the control (Fig. 7C); as with the other toxins, it became less complete at very positive potentials (above 0 mV). As found with toxins Shl and CgII, the slow component of inactivation induced by CpI decreased at higher potentials (Fig. 7A). The effects of the toxins on the midpoints for Na channel activation are summarized in Table 3. At 10°C, the shifts in activation were in general less than at the higher temperature, although there was considerable variation in this property. In the presence of these toxins the inactivation time course over part of the voltage range could be fit by a sum of two exponentials, as was previously shown for sodium currents in frog nodes of Ranvier, poisoned with scorpion a-toxin (WANG and STRIcELARTz, 1985) or the sea anemone toxin AsII (BERGMAN et al., 1976; UL.BRICHT and ScmmoTMAYER, 1981). There was a fast component with a time constant of 1-1 .5 cosec at 10°C and 0.5-0.9 cosec at 20°C (Table 4). This component decreased as the toxin effect became stronger . The slow component, as was evident in Figs 5, 6 and 7, had a time constant between 10 and 20 cosec for all three toxins, and gradually became smaller as pulse potential was made more positive, while the steady-state component became larger (Table 4). Depolarizing prepulses do not affect the current-voltage relation MmTs et al. (1982) found that depolarizing prepulses induced a transient shift in the

negative resistance branch of the sodium current-voltage relation to more negative potentials in the presence of scorpion (Centruroides) ß-type toxins . In our voltage clamp experiments at 10°C, we measured current-voltage relations in both the presence and

V. L. SALGADO and W. R. KEM

1376

control Cpl --D-- control, w/prepulse f Cpl, w/prepulse 10 MV

30 MV

14-S__offl v

Pro. 7. EFFBM or+ cA.L.ucnN (CPI) oN cRAYtuH ouxr AxoN . (A) Sodium current . (B) Peak Na current-voltage relation without (circles) and with (squares) a 30-msec propulse to +30 mV, ending 20 msec before each test pulse. (C) Steady-state Na inactivation for the same axon . Experiment V9607; temperature, 10°C .

absence of a 30 msec prepulse to + 30 mV, after a 20 msec recovery period at -100 mV. The only effect of this prepulse was to decrease the peak currents at all test potentials, due to increased inactivation; it did not shift the sodium channel current-voltage relation for any of the toxins . The current-voltage curves in control and Cpl-treated nerves are shown in Fig. 7B. Results with Shl and CgII were similar (data not shown) . Depolarization inhibits toxin action

When the axon membrane was held at a highly negative potential, the slowly inactivating current began to appear soon after perfusion of toxin Shl was initiated (Fig. 8, circles) ; after a small delay, the toxin effect appeared with a more-or-less exponential time course . In contrast, when another axon was depolarized to -65 mV during toxin application, the effect of Shl was minimal until the holding potential was returned to -110 mV (Fig. 8, squares) . At - 75 mV, the rate of toxin action was approximately halved (Fig. 8, triangles) . Similar results were obtained with CgII (not shown) .

Sea Anemone Toxins

1377

0.8 -1 0.6-9

0.4 -

Vhokt -

0 Fro. 8. ShI

moDmiicAnoN of

10

20

time

cawv>i

-110 MV-

30

AxoN soDnier cHANNE.a DEPOLARIZATION .

40min n

DmAYED By PAoLoN®

The circles show amplitude of the toxin-induced slow-inactivating current, normalized to peak current, for an experiment in which 100 nM ShI was added at -110 mV. The triangles and squares show normalized current amplitudes for experiments in which 100 nM ShI was added at holding potentials of -75 and -65 mV, respectively, for the period shown by the voltage protocol. The current was measured at the end of 2-meet test pulses to -10 mV, every 30 sec, each preceded by a 500-mwc conditioning hyperpolar zation to -110 mV to remove slow inactivation.

Strong depolarizations inhibit toxin delay of sodium channel inactivation

STiucHARTz and WANG (1986) found that strong depolarizing pulses caused Leiurus toxin to dissociate from Na channels in toad myelinated nerve. We carried out similar experiments with CgII and ShI, using 40 Hz pulses (data not shown) . Although the inactivation rates of the toxin-treated axons were accelerated during the repetitive depolarizations, there was also a large decrease ( > 50%) in peak sodium current which complicated interpretation of the results. This decrease in peak Na current could have been due to several causes, including a localized depletion of external Na concentration during the prolonged stimulation. HARTUNG and RATHMAYER (1985) also reported large reductions in the peak Na currents when AsH was investigated under repetitive pulse conditions. DISCUSSION

The major goal of this study was to compare the electrophysiological actions of a type 2 sea anemone toxin (ShI) with those of a type 1 toxin (CgII) on the same axon preparation. SCHWErlm et al. (1985) previously reported several types of evidence that these two types of toxin are pharmacologically as well as structurally distinct . We attempted to find differences in their electrophysiological actions, particularly since extensive voltage clamp analysis of pure crustacean-selective sea anemone toxins (mol. wt 5000) had not been

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previously carried out. Although the type 1 (actiniid) toxins have been rather extensively investigated by electrophysiological methods, only one voltage-clamp investigation of any type 2 anemone toxin had previously been reported . KRYSx'rAL et al. (1982) studied the effects of Heteractis (formerly Homostichanthus) macrodactylus toxin 1, a mammalian active type 2 toxin, upon cultured vertebrate neurons. The toxin selectively altered the inactivation properties of the tetrodotoxin-sensitive Na channel in a manner very similar to that found for a type 1 toxin, Anemonia sulcata toxin II (BERGMAN et al ., 1976). A single additional slowly inactivating Na current component was induced by the toxin. This new component failed to inactivate completely, even at a holding potential of 0 mV . This phenomenon, first reported by KOrmnm6FER and $CHMT (1968) for Leiurus (scorpion) venom action upon the frog node of Ranvier, was also observed for ASH action (BERGMAN et al., 1976). The membrane potential dependence of the faster, dominant Na channel population in the HmI-treated neuron displayed a steady-state inactivation relation that was not very different from the control inactivation curve, although the inactivation time constant was slightly increased. HmI did not perceptibly affect the voltage-dependence or kinetics of activation . One possible reported difference between type 2 (Hml) and type 1 (ASH) sea anemone toxin actions upon vertebrate neuronal Na channels concerns the change in the peak Na current: HmI nearly doubled the peak Na current in the study by KRYsHTAL et al. (1982), while the type 1 toxin ASII decreased the peak current in two other studies (BERGMAN et al., 1976 ; ULBRICHT and ScmmaDTMAYER, 1981). The effect of partially purified Condylactis toxin upon arthropod axons was to either slightly enhance the current (MuRAYAMA et al., 1972) or to double its size (PELHATE et al., 1976). In the present study, we found that all three crustacean toxins increased the peak Na current only in the negative resistance region of the current-voltage relation . Thus, at least with respect to crustacean neuron sodium channels, sea anemone toxins of all types have essentially identical effects on this phenomenon. GoNoi and HmLE (1987) pointed out that an irreversible voltage-independent inactivation model for the sodium channel, which fits data obtained in cultured neuroblastoma cells, predicts that slowing of inactivation would prolong the ascending phase of the Na current and cause a hyperpolarizing shift of the peak gNa-Em curve. At lower temperatures, the rate at which Na channels inactivate may be slow enough that most of the population of channels activated at a particular depolarizing potential will be open simultaneously, even when small depolarizing pulses are used . Under these conditions, the voltage dependence of peak sodium current would not be significantly affected by changes in inactivation caused by one of these toxins . Since increasing temperature increases the rate of inactivation more than the rate of activation, a greater indirect effect upon the activation (peak gNa vs . Em) curve by delaying inactivation would be expected at a higher temperature. For both ShI and CgII, the voltage dependence of the peak Na current was shifted to more negative potentials (Figs 5 and 6), as in several previous studies with toxins from sea anemones and scorpions (Low et al., 1979 ; GoNot and HILLE, 1987 ; HARTUNG and RATHMAYER, 1985). For both toxins, this shift was greater at 20°C than at 10°C (Table 3). CATTERALL and BfRESS (1978) found that binding of radioiodinated AsII and Mums toxin to vertebrate Na channels was inhibited by membrane depolarization, although the voltage dependence of ASII binding was less than that of Mums toxin binding. STRICHARTz and WANG (1986), using the voltage clamp technique, found that the toxinsuppressing effect of depolarization was not entirely explained by changes in toxin

Sea Anemone Toxins

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binding; they suggested that there is more than one toxin-bound state of the Na channel.

We attempted to assess whether the action of ShI could be influenced by conditioning pulses in a manner similar to type 1 sea anemone and scorpion a-toxins . Figure 8 shows that a depolarizing holding potential inhibited the onset of action of this toxin, in comparison to its onset of action at a more negative holding potential. The rate of toxin action was more than halved by depolarization to - 75 mV and almost completely inhibited by depolarization to -65 mV . The inhibition of toxin action by depolarization occurred at potentials considerably more negative than fast inactivation, which had a midpoint potential near -60 mV (Figs 5 and 7), but it is consistent with the voltage dependence for slow inactivation in the crayfish axon, which has a midpoint of -77 to -80 mV (SALGAno, 1992). Previous studies with Condylactis toxin utilized only partially purified samples that undoubtedly contained several neurotoxin variants (YOST and O'BRIEN, 1978 ; Ksm, in preparation) . Bi1tEss et al. (1976) isolated four neurotoxins from the related European sea anemone Condylactis aurantica. In addition, Condylactis tentacular acetone powders also contain cytolytic protein toxins (BERNHEmER and AviGAD, 1981 ; KEm, unpublished results) . Nevertheless, the results of these earlier studies are in fundamental agreement with ours using pure CgII, with one possible exception: MuRAYAMA et al. (1972) and PELHATE et aL (1976) reported that the K channel current was sometimes depressed as much as 20% . We did not observe any depression of the K current. It is possible that Condylactis toxin extracts also contain toxins affecting K channels, as is the case for many scorpion venoms. Alternatively, there may have been a decrease in axonal potassium concentration in the earlier investigations, where the axons were not internally perfused. The irreversibility of sea anemone toxin action upon crustacean Na channels has been consistently observed by all investigators, whereas toxin effects on vertebrate or insect neurons are readily reversible . The difference between the sea anemone toxins regarding their crustacean, insect, and mammalian toxicity are quite large (Table 1) . We recently observed similar differences in the ability of ShI to bind to neuronal membranes of crab and rat (PENNINGToN et al., unpublished results) . Also, NARAHmm et al. (1969) found that Condylactis toxin had no effect on molluscan (squid) neuron Na channels . A crustaceanselective scorpion toxin selectively inhibits Na channel inactivation (ZLoTK.IN et al., 1975 ; RATHmAYER et al., 1977; PELHATE and Zc.oT1c1N, 1982). These phyletic differences in toxin activity are intriguing and deserve further study by electrophysiological, ligand-binding, and molecular biological methods. Further comparisons of electrophysiological effects and binding properties between the various types of sea anemone toxins are necessary to rigorously test the hypothesis that the toxins have more than one binding site on the Na channel and mechanism of action. In contrast with the previous reported differences in membrane binding, etc., between types 1 and 2 sea anemone toxins, our study has revealed remarkable similarity in the effects of ShI, CgII and CpI on the crayfish sodium channel. However, there may be some small quantitative differences . For instance, Shl apparently has a greater ability to depolarize the resting membrane and produce a longer duration plateau in the repolarization phase of the action potential, compared to CgII . The depolarizing effect could have resulted from opening only a small fraction of the Na channels present in the axon, as has been observed for toxins producing persistent activation of Na channels (STRICHARTz et al., 1987). These differences may still be explainable on the basis of a single receptor site for sea anemone and scorpion at-toxins. Patch-clamp experiments at the single Na channel

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level (NAGY, 1988) should be able to detect subtle differences in the mechanism of action of these structually diverse sea anemone toxins . Alawwledgemrats-In addition to the facilities at Rohm and Haas Company, this research was partially funded by NIH (GM 32848) and NATO (0123,87) grants to W . KEm . We are grateful to DR Lucto CARaLLo (Zoological Station, Naples, Italy), for providing pure calitoxin (Cpl) and to Ms JuDY ADAM for word processing of the manuscript.

REFERENCES Bftm, R, Bimms, L. and WuNDERmt, G. (1976) Purification and characteriza tion of four polypeptides with neurotoxic activity from Condylactis auranticw . Hoppe-Seyler's Z. physiol. Chem . 357, 409-614. BattauN, C ., DuBois, J. M., Roses, E. and RATHuAYER, W. (1976) Decreased rate of sodium conductance inactivation in the node of Ranvier induced by a polypeptide toxin from sea anemone. Biochlm . biophys. Acts 455,173-184. BmeriiHma, A. W. and AvtoAD, L . S. (1981) New cytolysins in sea anemones from the west coast of the United States. Toxicon 19, 529-534 . CARna.LO, L ., DE SANTis, A., FSoRE, F., Ptocou, R., $PAoNuoLo, A ., ZANtrTn, L. and PARIiNTE, A . (1989) Calitoxin, a neurotoxic peptide from the sea anemone Cafactis parasitica: amino acid sequence and electrophysiological Properties. Biochemistry 28, 2484-2489. CArrERAut, W. A. and Bbms, L. (1978) Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium iontophore . J. biol. Chem . 233, 7393-7396. CATrERALL, W. A. and Copmtnam J. (1981) Pharmacological properties of sodium channels in cultured in rat heart cells . Mole Pharmac. 20, 533-541 . GoNa, T. and HIua, B. (1987) Inactivation modifiers discriminate among models . J. gene Physiol. 99,253-274. GoNot, T., Hmu, B . and CArnm"Lr , W . A. (1984) Voltage clamp analysis of sodium channels in normal and scorpion toxin-resistant neuroblastoma cells. J. Neurosci. 4, 2836-2842. HARTuNo, K. and RATtbuymy W . (1985) Ammonia sulcata toxins modify activation and inactivation of Na+ currents in a crayfish neurone . Piers Arch . ges. Physiol. 404, 119-125 . KESC, W. R (1988x) Sea anemone toxin structure and action . In : The Biology of Nematocysts, pp. 375-405 (HmsQtomr, D . and Ltnas mp, H., Eds) . New York: Academic Press. Kms, W . R . (1988b) Peptide chain toxins of marine animals . In: Biomedical Importance of Marine Organisms (FAunN, D ., Ed.) Mere. Calf. Acad. Sci. 13, 69-83. Kim, W . R., PmtTEN, B ., PENmmToN, M . W., Paid, D . A. and Dtnvw, B . M . (1989) Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthra . Biochemistry 28, 3483-3489 . KoppSNHbmt, E . and ScrmenT, H . (1968) Incomplete sodium inactivation in nodes of Ranvier treated with scorpion venom . Experientia 13, 41-42 . KxystuAL, G. A ., Osmsut