Anemonia sulcata toxins modify activation and inactivation of Na+ currents in a crayfish neurone

1. The effects of three toxins (ATX I, II, III) isolated from the sea anemoneAnemonia sulcata were studied in the soma membrane of a crustacean neurone under voltage-clamp conditions. 2. All three toxins affected the action potentials and the Na⁺ currents in a similar manner. The lowest concentrations tested (10 nM, 20 nM and 50 nM for AtX I, II and III, respectively) had pronounced selective effects on the Na⁺ current. No effect on K⁺ or Ca²⁺ currents was observed with concentrations up to 5 M. 3. In the presence of ATX the Na⁺ inactivation was incomplete even with pulses of 700 ms length or strong depolarizing prepulses. 4. Besides the effects on the inactivation process ATX affected also the activation of the Na⁺ current. 5. In cells treated with ATX the negative resistance branch of the peak Na⁺ current voltage relation was shifted by –5 mV to –20 mV. 6. The time to peak was increased for small depolarizations (up to –30 mV) and the rate of rise (I/t) was enlarged by ATX. A slow activating current component was also observed after depolarizing prepulses or if the Na⁺ current was outward. 7. The decay of the Na⁺ tail currents was considerably prolonged after the application of ATX if the membrane was repolarized to potentials more positive than about –60 mV. 8. Repetitive stimulation led to a shortening of the action potential in ATX II treated neurones. A simultaneous and parallel decrement of the peak and plateau current was observed with depolarizing voltage steps.     

Differences in the binding sites of two site-3 sodium channel toxins

 Site-3 toxins from scorpion and sea anemone bind to Na channels and selectively inhibit current decay. Anthopleurins A and B (ApA and ApB, respectively), toxins found in the venom of the sea anemone Anthopleura xanthogrammica, bind to closed states of mammalian skeletal and cardiac Na channels with differing affinities which arise from differences in first-order toxin/channel dissociation rate constants, k _off. Using chimera comprising domain interchanges between channel isoforms, we examined the structural basis of this differential affinity. Toxin/channel association rates, k _on, were similar for both toxins and both parental channels. Domain 4 determined k _off for ApA, while ApB dissociated from all tested chimera in a cardiac-like manner. To probe this surprising difference between two such closely related toxins, we examined the interaction of chimeric channels with a form of ApB in which the two nonconserved basic residues, Arg-12 and Lys-49, were converted to the corresponding neutral amino acids from ApA. In the chimera comprising domain 1 from the cardiac muscle isoform and domains 2–4 from the skeletal muscle isoform, toxin dissociated at a rate intermediate between those of the parental channels. We conclude that the differential component of ApA binding is controlled by domain 4 and that some component of ApB binding is not shared by ApA. This additional component probably binds to an interface between channel domains and is partly mediated by toxin residues Arg-12 and Lys-49. Received: 17 April 1997 / Received after revision: 20 June 1997 / Accepted: 26 June 1997     

Changes in Na channel properties of frog and rat skeletal muscles induced by the AaH II toxin from the scorpion Androctonus australis

The effects of the mammal toxin II isolated from the venom of the scorpion Androctonus australis Hector (AaH II) were studied under current and voltage clamp conditions in frog (semitendinosus) and rat (fast e.d.l. and slow soleus) skeletal twitch muscle fibres. In both species, AaH II induced a dose-dependent prolongation of the action potential (AP) leading at saturating concentration to APs with long plateaus of about 1.5 s in frog and 5 s in rat e.d.l. and soleus fibres. The concentrations to induce 50% of the maximal effect (K _0.5) were 9.1×10^–9 M in the frog and 1.4×10^–9 M in the rat. AaH II increased the time constants of inactivation of the peak Na current and induced a maintained Na current that was greater in rat e.d.l. and soleus (31.6 % of peak current amplitude at — 30 mV; K _0.5= 0.8×10^–9M) than in frog (16.5%; K _0.5=15.5 ×10^–9 M) muscles. Peak and maintained Na currents were TTX-sensitive and had identical threshold and reversal potentials. The half-maximum maintained permeability occurred at a potential 20 mV more positive than the peak permeability. Recovery from inactivation and steady-state inactivation of the inactivating Na current remained unchanged. The maintained current deactivated with normal fast kinetics. The action of the toxin reversed poorly on washout but could be largely removed by conditioning depolarizations more positive than the reversal potential of the Na current. Our results suggest that, in vertebrate skeletal muscle fibres, AaH II affects all the Na channels and are consistent with the hypothesis that the maintained current originates from a reopening of previously inactivated Na channels.     

Effect of toxins isolated from the venom of the scorpionCentruroides sculpturatus on the Na currents of the node of ranvier

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).     

Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh II, Lqh III, and LqhαIT

The scorpion alpha-toxins Lqh II, Lqh III, and Lqh alphaIT from Leiurus quinquestriatus hebraeus are representatives of typical alpha-toxins, specific for either mammals (Lqh II) or insects (Lqh alphaIT), and alpha-like toxins (Lqh III) which act on both mammals and insects. For a comparative study of the effects of these toxins on mammalian sodium channels we stably expressed rat skeletal muscle sodium channel alpha subunits (microI) in HEK 293 cells and measured Na+ currents in the whole-cell patch-clamp mode. The alpha- and alpha-like toxins strongly slowed down channel inactivation with a half-maximal effect at 1.4 nM (Lqh II), 5.4 nM (Lqh III), and 0.5 nM (Lqh alphaIT). The recovery from fast inactivation was accelerated by all toxins with the potency sequence: Lqh II>Lqh alphaIT>Lqh III. The voltage dependence of inactivation and recovery from inactivation were reduced while the threshold for activation was only slightly shifted by approximately equal to 10 mV without altering the slope factors, suggesting uncoupling of the impaired inactivation from the activation. The toxins induced an increase in peak inward current, which was accounted for by an increased maximal open-channel probability. Although all three toxins induced similar modifications of the channel properties, their kinetics of association and dissociation were very different. Between -140 and -80 mV toxin association was not voltage dependent. In 100 nM toxin the association time constants were: 1.3 s (Lqh II), 20 s (Lqh III), and 3.8 s (Lqh alphaIT). At positive voltages the toxin dissociated from the channel; at +100 mV the dissociation time constants were 30, 321, and 135 ms, respectively. In contrast to the association, dissociation was voltage dependent with a similar slope of about 12 mV per e-fold change for all three toxins. The strong differences in the association and dissociation kinetics of these toxins may identify them as members of different scorpion alpha-toxin subgroups.     

Kinetics and distribution of voltage-gated Ca,Na and K channels on the somata of rat cerebellar Purkinje cells

Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels.     

Voltage-dependent effect of a scorpion toxin on sodium current inactivation

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.     

A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1

The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives were found to be biologically active. In electrophysiological patch-clamp recordings mono-[¹²⁷I]-BeKm-1 had a concentration of half-maximal inhibition (IC₅₀ value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC₅₀ value of 7 nM. Mono-[¹²⁵I]-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles from transfected human embryonic kidney cells (HEK-293). Under optimized conditions the equilibrium dissociation constant (K_d) values from saturation and kinetic binding analysis were 13 and 14 pM, respectively. Both the association and dissociation of [¹²⁵I]-BeKm-1 were fast (association rate constant, k_on=3.6×10⁷ M^–1s^–1; dissociation rate constant, k_off=0.005 s^–1). Wild-type BeKm-1 displaced binding of [¹²⁵I]-BeKm-1 with half-maximal inhibitory concentrations of 44 pM. In contrast, competition experiments with a BeKm-1 mutant BeKm-1-K18A, in which the toxin interaction site is disrupted, resulted in a drop in affinity by more than 300-fold as compared to the wild-type toxin. Iberiotoxin and apamin, peptide inhibitors of Ca²⁺-activated K⁺-channels, had no effect on [¹²⁵I]-BeKm-1 binding. Adding the classical rapid delayed rectifier current (I_Kr) blocker E-4031 reduced binding of [¹²⁵I]-BeKm-1 to the hERG channel to an IC₅₀ of 7 nM. In autoradiographic studies on rat hearts, binding of [¹²⁵I]-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed in the myocardium of both the ventricle and atria indicating a homogenous expression of hERG channels throughout the heart.     

Frequency-dependent effects of aconitine and veratridine on membrane currents in the crayfish giant axon

Effects of aconitine and veratridine on membrane currents in the crayfish giant axon were studied under voltage-clamped conditions. Aconitine at 50 microM reduced the Na current without changing the K current. The Na current left after aconitine block showed the activation and inactivation kinetics unaltered from the normal. These aconitine effects differ from those reported previously with myelinated nerves. The rate of Na channel block by aconitine was increased with increasing either frequency or voltage of depolarizing pulses delivered repetitively from the holding potential but was not affected by a change in the pulse duration from 2 to 10 msec. The same change in the pulse duration, however, caused about a 4-fold increase in the blocking rate in the axon of which inactivation process had been removed by a pretreatment with a sea anemone toxin from Parasicyonis actinostoloides. These results are explained by a model in which Na channels are occluded with aconitine molecules only when the channels are open. Veratridine at 50 microM also exhibited frequency-dependent actions, depressing the maximum peak inward current and shifting the reversal potential of the transient current in the hyperpolarized direction. These veratridine effects persisted after washing. In addition to such persistent actions, veratridine induced a maintained Na current at the holding potential during repetitive stimulation. This effect was abolished after a brief wash unlike the above-mentioned persistent effects, suggesting that veratridine has two (or more) different modes of actions on Na channels in the crayfish giant axon.     

Action of tetanus toxin and colchicine on synaptic membranes of the rat cerebral cortex

Purified tetanus toxin (TT), in experiments in vitro, was shown to affect neither the Na,K-ATPase activity of the synaptic membrane fraction of the rat cerebral cortex nor the inhibition of Na,K-ATPase activity produced by electrical stimulation of a suspension of synaptic membranes, nor the binding of GABA-³H by synaptosomes. TT and colchicine (1 mM) reduced the osmotic sensitivity of the nerve endings. Colchicine, in low concentrations (10^–5 to 10^–3 M), does not affect Mg- and Na,K-ATPase but, in higher concentrations (10^–2 M), it inhibits the activity of both ATPases considerably.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 139–142, February, 1977.     

The effect ofTityus serrulatus scorpion toxin γ on Na channels in neuroblastoma cells

1. The effect of highly purified toxin γ from the venom of the scorpionTityus serrulatus (TiTxγ) on nerve membrane ionic channels have been investigated using the suction electrodes voltage clamp technique on neuroblastoma cells.
2. The amplitude of the normally voltage-dependent Na current is reversible reduced by approximately 50% after 15–105 nM TiTxγ, whereas even the highest toxin concentrations have no significant effect on the outward K current in the presence of tetrodotoxin.
3. TiTxγ causes a transient inward current to appear at membrane potentials between ?70 and ?40 mV, a potential region in which no significant inward current is observed in control experiments.
4. Tetrodotoxin (300 nM) rapidly blocks both the TiTxγ-induced inward current and the remaining normally voltagedependent Na current. The binding of radiolabelled TiTxγ to the Na channels in the neuroblastoma cell membrane is prevented by native TiTxγ with aK _0.5=0.75 nM.
5. Both activation and inactivation of the TiTxγ-induced Na current are shifted 30–40 mV towards more negative potential values as compared to normally voltage-dependent Na current. The TiTxγ-induced Na current exhibits sigmoidal activation kinetics and relatively slow, exponential inactivation kinetics.
6. The local anesthetic procaine at an external concentration of 1 mM blocks more effectively the remaining normally voltage-dependent Na current than the TiTxγ-induced Na current. Both Na current components are equally blocked by 1 mM of the local anesthetic propoxycaine.
7. The relation between the effects of TiTxγ on Na⁺ channels and those of other known neurotoxins specific of this channel is discussed. It is concluded that the characteristic effects of TiTxγ differ from those of all other known toxins including other scorpion toxins that bind to the same site on the Na channel.

     

Evidence for two calcium currents in insulin-secreting cells

Inward currents were measured in cultured rat pancreatic islet cells using the whole-cell patch-clamp technique. Depolarization elicited both transient and more sustained inward currents. The transient current was blocked by external Na removal, TTX or depolarizing holding potentials while the sustained current was blocked by external Ca removal or the addition of Co, Ni or Cd ions to the external solution. Replacing Ca with Ba increased the amplitude of the sustained current and decreased its rate of decay. These results suggest that the transient current was due to the activation of Na channels while the sustained current was mediated by Ca channels. Two characteristics of the data suggested that two Ca currents may contribute to the sustained current. First, a second shoulder, or hump, was observed on the descending limb of the Ca current I–V of many cells, suggesting that they may possess two Ca channels that activate at different voltages. Second, the low and high voltage components of the I–V were differentially suppressed by Cd. Neither of the Ca current components was very sensitive to the Ca channel agonist BAY K 8644. These results were confirmed in HIT cells, another insulinsecreting preparation.     

Gating current experiments on frog nodes of Ranvier treated withCentruroides sculpturatus toxins or aconitine

(1) Gating currents were recorded from frog nodes of Ranvier treated either with toxins III or IV from the venom of the scorpionCentruroides sculpturatus or with the alkaloid toxin aconitine. (2) Toxins III or IV fromCentruroides sculpturatus (which drastically reduce the sodium permeabilityP _Na and slightly shift its voltage dependence in the depolarizing direction) caused a small depolarizing shift of the relation between charge (Q _on) and membrane potential (E) without affecting the maximum chargeQ _{on max}. (3) On nodes treated with toxins III or IV fromCentruroides sculpturatus, a depolarizing conditioning pulse (which transiently shifts the descending branch of theI _Na(E) curve by up to 60 mV in the hyperpolarizing direction) shifted the midpoint potential (E_mid) of theQ _on(E) curve by –17 mV and slightly increased the slope of the curve; it also decreasedQ _{on max} markedly but had little effect onQ _on 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 theQ _on(E) curve from –28.5 to –69 mV and significantly increasedQ _on andQ _off measured with small depolarizing pulses; concomitantly, the voltage dependence of the on time constant of the charge movement [_on(E)] was shifted by –44 mV. (4) The sodium currentI _Na was exponential both in nodes treated with toxins III or IV ofCentruroides sculpturatus and subjected to a depolarizing conditioning pulse and in aconitine-treated nodes; in the latter,I _Na started after a delay of 30–40 s. The time constant of the sodium current, _{on Na}, was larger than the time constant of the charge movement, _{on Q}; the ratio _{on Q}/_{on Na} was 0.61 and 0.73 in the experiments withCentruroides sculpturatus toxins and aconitine, respectively. (5) The off time constant of the sodium current (_{off Na}) was slightly increased in nodes treated withCentruroides 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 (_{off Q}) was unaffected but with aconitine treatment it was considerably increased although it remained smaller than _{off Na}. Consequently, the ratio _{off Q}/_{off Na} (which is 1 in untreated nodes) became smaller than one, reaching values as low as 0.58 and 0.44 in the experiments withCentruroides sculpturatus toxins and aconitine, respectively. The small _{off Q}/_{off Na} ratio suggests that the channels remain open for an appreciable time after most of the gating charges have returned to their resting position. (6) The results obtained with aconitine resemble the findings on batrachotoxin-treated nodes (Dubois and Schneider 1985), except that in the latter the time constants _{on Na} and _{off Na} of the sodium current are smaller than the corresponding time constants _{on Q} and _{off Q} of the charge movement.     

Effects of toxins VI and VII from the scorpionCentruroides sculpturatus on the Na currents of the frog node of Ranvier

1. New toxins, VI and VII, were purified from the venom of the North American scorption,Centruroides sculpturatus, and studied in the voltage-clamped frog node of Ranvier. These toxins reduced peak inward Na currents and caused a transient, depolarization-induced shift in the voltage dependence of Na activation. Their effects were indistinguishable from those of toxins I, III and IV, as previously described by Meves et al. (1982). 2. Toxin VII, 0.2 g/ml, shifted the voltage dependence of steady-state inactivation (h ). In four fibers, the mean shift of theh (E) curve was –17 mV, compared to a mean shift of –28 mV in the descending branch of theI _Na(E) curve. Theh (E) curve with toxin VII was monotonic and inactivation was incomplete at positive potentials. 3. Decreasing the pH from 7.4 to 5.7 increased the shift in the voltage dependence of activation with toxin. The increase with toxin VI at pH 5.7 was reversible on returning to pH 7.4, but the increase with toxin VII was not. The lack of reversibility of the effect of pH with toxin VII was quantified in two ways: (a) the mean value ofI _Na (measured at –62 mV with a conditioning pulse) at pH 7.4 was 5.1 times larger after treatment than before treatment with 0.1 g/ml of toxin VII at pH 5.7; (b) the average concentration of toxin VII required for a shift of –30 mV at pH 7.4 was decreased by a factor of 4.3, to 0.19 g/ml (26 nM), by pretreatment at pH 5.7. In comparison, a shift of –30 mV was produced with 2.31 g/ml (319 nM) of toxin VI. 4. For both toxin VI and toxin VII (in the latter case, after initial pretreatment with toxin at pH 5.7), the shift in the voltage dependence of activation with a conditioning pulse was 1.8 times larger at pH 5.7 than at pH 7.4. The increase was accounted for by an essentially normal shift (mean = +13.1 mV;n=7) of theI _Na(E) curve without a conditioning pulse at pH 5.7, but a minimal shift (mean = +0.4 mV) of theI _Na(E) curve with a conditioning pulse. Permeability ratios at the two pH's reached the value of 0.5 at a membrane potential of +28 mV without a conditioning pulse, whereas this value was reached near –62 mV when a conditioning pulse preceded the test pulse. 5. These observations were interpreted as evidence that the binding of both toxin VI and toxin VII is greater at pH 5.7 than at pH 7.4, but that the increase with toxin VI is reversible on increasing the pH whereas the increase with toxin VII is not under the experimental conditions used.     

Cytotoxic effects by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence ofClostridium perfringens iota toxin

The ADP-ribosylating toxinsClostridium botulinum C2 toxin andC. perfringens iota toxin, which ADP-ribosylate monomeric G-actin at Arg-177 but not the polymeric F-actin, induce depolymerization of the actin cytoskeleton in cultured cells. Since ADP-ribosylated G-actin has properties of a barbed-end-capping protein, we studied whether the ADP-ribosylated actin affects the actin cytoskeleton of PtK2 cells even in the absence of ADP-ribosylating toxin. Skeletal muscle actin was ADP-ribosylated byC. perfringens iota toxin and the toxin was removed using an anti-iota toxin antibody. Microinjection of ADP-ribosylated actin caused retraction of the cell body, redistribution and depolymerization of the actin cytoskeleton in a concentration-and time-dependent manner. The finding that ADP-ribosylated actin affects per se the actin cytoskeleton explains the cytopathic effects of ADP-ribosylating toxins on microfilaments, although F-actin is not directly modified by the toxins.     

Poneratoxin,a new toxin from an ant venom,reveals an interconversion between two gating modes of the Na channels in frog skeletal muscle fibres

The effects of synthetic poneratoxin (PoTX), a new toxin isolated from the venom of the ant Paraponera clavata, were studied under current- and voltage-clamp conditions in frog skeletal muscle fibres. PoTX induces a concentration-dependent (10^–9 M–5×10^–6 M) prolongation of the action potentials and, at saturating concentration, a slow repetitive activity developing at negative potentials. PoTX specifically acts on voltage-dependent Na channels by decreasing the peak Na current (I _Na) and by simultaneously inducing a slow I _Na which starts to activate at –85 mV and inactivates very slowly. Both the fast and the slow components of I _Na are suppressed by tetrodotoxin and reverse at the same potential corresponding to the equilibrium potential for Na ions. The fast component of I _Na has voltage dependence, activation and steady-state inactivation almost similar to those of the control I _Na. The voltage dependence of the slow Na conductance is 40 mV more negative than that of the fast one. The results suggest that PoTX affects all the Na channels and that the fast and the slow I _Na components originate from a possible PoTX-induced interconversion between a fast and a slow operating mode of the Na channels.     

Potential-dependent effects of sea anemone toxins and scorpion venom on crayfish giant axon

Effects of two kinds of sea anemone toxin (Parasicyonis actinostoloides andAnemonia sulcata) and scorpion venom (Leiurus quinquestriatus) on crayfish giant axons were examined electrophysiologically. All toxins acted on the axon in a similar manner to prolong the falling phase of the action potential. In all cases the development of toxicity was reduced when the nerve membrane was depolarized by a current injection. However, the ranges of membrane potential where the significant reduction in toxicity took place were different for each toxin. The action of Parasicyonis toxin was also suppressed by depolarization resulting from treatment of the axon with a neurotoxic alkaloid, veratridine. The mechanism of the potential-dependent toxin action is discussed with reference to the present data and relevant works by other investigators.     

Modification of sodium channel gating and kinetics by versutoxin from the Australian funnel-web spiderHadronyche versuta

The effects of a neurotoxin (versutoxin VTX), purified from the venom of the Australian Blue Mountains funnel-web spiderHadronyche versuta, on the ionic currents in rat dorsal root ganglion cells were investigated under voltage-clamp conditions using the whole-cell patch-clamp technique. VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. In contrast VTX produced a dosedependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation, a reduction in peak TTX-S sodium current but did not markedly slow tail current kinetics of TTX-S sodium currents. This steady-state sodium current was maintained during prolonged depolarizations at all test potentials and the reduction in sodium current amplitude produced by VTX had an apparentK _i of 37 nM. In the presence of 32 nM VTX the voltage dependence of steady-state sodium channel inactivation (h ) also showed a significant 7 mV shift in the voltage midpoint in the hyperpolarizing direction, with no change in the slope factor. In addition there was a steady-state or non-inactivating component present (14±2% of maximal sodium current) at prepulse potentials more depolarized than –40 mV, potentials which normally inactivate all TTX-S sodium channels. Finally, there was an observed increase in the rate of recovery from inactivation in the presence of VTX. These selective actions of VTX on sodium channel gating and kinetics are similar to those of-scorpion and sea anemone toxins.     

Identification of specific binding sites for nerve growth factor on human blood platelets and membranes from bovine brain

The presence of specific binding sites for nerve growth factor on membranes from bovine brain and human blood platelets is shown. The association and dissociation kinetics and the dissociation constants of receptor binding of nerve growth factor are analyzed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 295–297, March, 1996 Presented by N. P. Bochkov, Member of the Russian Academy of Medical Sciences     

Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation

 The present study investigates the actions of robustoxin (atracotoxin-Ar1) purified from the venom of the male Sydney funnel-web spider Atrax robustus on sodium channel gating. Using whole-cell patch-clamp techniques the study assessed the actions of robustoxin on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in rat dorsal root ganglion cells. Similar to the closely related funnel-web spider toxin versutoxin (δ-atracotoxin-Hv1) from Hadronyche versuta, robustoxin had no effect on TTX-R sodium currents but exerted potent effects on TTX-S sodium currents. The main action of robustoxin was a concentration-dependent slowing or removal of TTX-S sodium current inactivation. This steady-state current was maintained during long-lasting depolarisations at all test potentials. Robustoxin (30 nM) also caused a 13-mV hyperpolarising shift in the voltage midpoint of steady-state sodium channel inactivation (h _∞) leading to a reduced peak current at a holding potential of –80 mV. Moreover there was a steady-state or non-inactivating component present (18% of maximal sodium current) at prepulse potentials that normally inactivate all TTX-S sodium channels (more depolarised than –40 mV). In addition robustoxin produced a significant increase in the repriming kinetics of the sodium channel when channels returned to the resting state following activation. This increase in the rate of recovery of sodium current appears to explain the use-dependent effects on peak sodium current amplitude at high stimulation frequencies. Finally 30 nM robustoxin caused an 11-mV hyperpolarising shift in the voltage dependence of the channel but did not markedly modify tail current kinetics. These actions suggest that robustoxin inhibits conversion of the open state to the inactivated state of TTX-S sodium channels, thus allowing a fraction of the sodium current to remain at membrane potentials at which inactivation is normally complete. Given the recent reclassification of funnel-web spider toxins as atracotoxins, robustoxin should henceforth be known as δ-atracotoxin-Ar1 to reflect this main action on sodium channel inactivation. These present results further support the hypothesis that funnel-web spider toxins interact with neurotoxin receptor site 3 to slow channel inactivation in a manner similar to that of α-scorpion and sea anemone toxins. Received: 9 September 1997/Received after revision: 15 January 1998/Accepted: 29 January 1998