Preface

The physiological function of the axon is to conduct short all-or-none action potentials from their site of initiation (usually the cell body) to the synapse. To ensure this function, both passive and active biophysical properties of the axons are tuned very precisely, especially the voltage-dependent ionic conductances to sodium and potassium. Under normal conditions, axons are not spontaneously active. Minor modifications of their ionic micro-environment or slight changes in the membrane properties are however sufficient to induce rhythmical activity and modify the time course of the action potentials. These modifications can be induced by a variety of pharmacological agents. Some typical examples taken from original studies on invertebrate preparations are illustrated. The experiments were carried out on two axonal preparations: the giant axon of the squid Loligo forbesi and the giant axon of the cockroach Periplaneta americana. The axons were ‘space-clamped’ and studied under both current-clamp and voltage-clamp conditions. Voltage-clamp experiments were used to dissect out the mechanisms underlying repetitive activity and to extract the relevant parameters. These parameters were then used to rebuild the observed effects using an extended version of the Hodgkin and Huxley (1952, J Physiol (Lond) 117, 500–544) formulation. One easy way to get repetitive firing in both preparations is to reduce potassium conductance. The effect of 4-aminopyridine on squid axon is illustrated here. The experimental results, including the occurrence of bursts of activity, can be described by adding a time- and voltage-dependent block of the potassium channels to the original Hodgkin and Huxley (1952, J Physiol (Lond) 117, 500–544) model. Repetitive spike activity and plateau action potentials are also produced when the depolarising effect of the voltage-dependent potassium current is counterbalanced by a maintained inward sodium current. This maintained sodium current can be due to several different mechanisms. This will be illustrated by five structurally unrelated molecules: two scorpion toxins, two insecticide molecules and one sea anemone toxin. One toxin purified from the venom of the scorpion Buthotus judaïcus (insect toxin 1) exerts its effects by shifting the sodium activation curve towards more hyperpolarized potentials. Another toxin purified from the venom of another scorption Androctonus australis (mammal toxin 1) modifies a significant proportion of normal (fast) sodium channels into slowly activating and inactivating sodium channels. The main effect of the insecticide DDT is to maintain sodium channels in the ‘open’ configuration. Another insecticide molecule known to induce repetitive activity, S-bioallethrin, activates voltage-dependent sodium channels with slow activation and inactivation kinetics. The sea anemone toxin anthopleurin A, purified from the venom of Anthopleura xanthogrammica, delays inactivation of the sodium current without changing its activation kinetics. These examples show that minor modifications of the properties of the nerve membrane are sufficient to alter nerve function. These deleterious effects will be amplified at the synapse through dramatic changes in transmitter release and will lead eventually to disastrous alterations of brain function.     

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site

Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore.

Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.     

Effect of toxin 1 from Androctonus australis Hector on sodium currents in giant axons of Loligo forbesi

The effects of external application of micromolar concentrations of toxin 1 of the scorpion, Androctonus australis Hector, on the sodium conductance of squid giant axons have been studied quantitatively using the voltage clamp technique. Toxin concentrations which induce long plateau action potentials under current clamp conditions were found to simultaneously decrease the peak conductance and increase the delayed sodium conductance. Return to holding potential level after step depolarizations was accompanied by large exponential tails of current. The toxin-induced maintained sodium conductance increased with membrane depolarization independently of the peak conductance. Depolarizing conditioning prepulses to - 30 mV were found to almost totally inactivate the peak sodium current but to leave the delayed conductance unaffected. This property was taken as an indication that the total current is made of the added contributions of two distinct populations on sodium channels : fast activating and inactivating channels and slow activating channels. These two channel populations were separated from each other and analysed. It was found that the fast channels were almost identical to normal channels whereas the slow channels had a much slower (nearly exponential) kinetics and activated for more positive values of membrane potential. These observations strongly support the second hypothesis of Gillespie and Meves (1980) that the peak conductance and maintained conductance reflect the existence of two separate populations of channels. They further indicate that slow channels probably originate from the modification by the toxin of normal voltage-sensitive channels.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Rapid voltage-dependent dissociation of scorpion alpha-toxins coupled to Na channel inactivation in amphibian myelinated nerves

The voltage-dependent action of several scorpion alpha-toxins on Na channels was studied in toad myelinated nerve under voltage clamp. These toxins slow the declining phase of macroscopic Na current, apparently by inhibiting an irreversible channel inactivation step and thus permitting channels to reopen from a closed state in depolarized membranes. In this article, we describe the rapid reversal of alpha-toxin action by membrane depolarizations more positive than +20 mV, an effect not achieved by extensive washing. Depolarizations that were increasingly positive and of longer duration caused the toxin to dissociate faster and more completely, but only up to a limiting extent. Repetitive pulses had a cumulative effect equal to that of a single pulse lasting as long as their combined duration. When the membrane of a nonperfused fiber was repolarized, the effects of the toxin returned completely, but if the fiber was perfused during the conditioning procedure, recovery was incomplete and occurred more slowly, as it did at lower applied toxin concentrations. Other alpha-type toxins, from the scorpion Centruroides sculpturatus (IVa) and the sea anemone Anemonia sulcata (ATXII), exhibited similar voltage-dependent binding, though each had its own voltage range and dissociation rate. We suggest that the dissociation of the toxin molecule from the Na channel is coupled to the inactivation process. An equivalent valence for inactivation gating, of less than 1 e per channel, is calculated from the voltage-dependent change in toxin affinity.     

The sodium channel in non-impulsive cells Interaction with specific neurotoxins

The cell line C₉ used in this paper has a resting potential of ?50 mV (±10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na⁺ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstrated by electrophysiological techniques and by ²²Na⁺ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. ²²Na⁺ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of ²²Na⁺ influx into the C₉ cell. The stimulation of ²²Na⁺ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of ²²Na⁺ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Simultaneous modifications of sodium channel gating by two scorpion toxins.

The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan (1975, J. Physiol. [Lond.]. 244:511-534) for the crude scorpion venom. Both of these effects were observed in fibers treated with both of these toxins, and the kinetics of the induced current were modified in a way that showed that the same sodium channels were modified simultaneously by both toxins. Although the toxins can act on different sites, the time course of the action of C. sculpturatus toxin was accelerated in the presence of the L. quinquestriatus toxin, indicating some form of interaction between the two toxin binding sites.     

Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel

Four new toxins have been isolated from the sea anemone Radianthus paumotensis: RpI, RpII, RpIII, and RpIV. They are polypeptides comprised of 48 or 49 amino acids; the sequence of RpII has been determined. Toxicities of these toxins in mice and crabs are similar to those of the other known sea anemone toxins, but they fall into a different immunochemically defined class. The sequence of RpII shows close similarities with the N-terminal end (up to residue 20) of the previously sequenced long sea anemone toxins, but most of the remaining part of the molecule is completely different. Like the other sea anemone toxins, Radianthus toxins are active on sodium channels; they slow down the inactivation process. Through their Na+ channel action, Radianthus toxins stimulate Na+ influx into tetrodotoxin-sensitive neuroblastoma cells and tetrodotoxin-resistant rat skeletal myoblasts. The efficiency of the toxins is similar in the two cellular systems. In that respect, Radianthus toxins behave much more like scorpion neurotoxins than sea anemone toxins from Anemonia sulcata or Anthopleura xanthogrammica. In binding experiments to synaptosomal Na+ channels, Radianthus toxins compete with toxin II from the scorpion Androctonus australis but not with toxins II and V from Anemonia sulcata.     

Functional duality and structural uniqueness of depressant insect-selective neurotoxins

Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.     

Effects of tetraethylammonium and 4-aminopyridine on the plateau potential of circular myometrium from the pregnant rat

In the pregnant rat, spontaneous electrical activity of circular muscle (CM) changes from single, plateau-type action potentials at early and mid-term to repetitive spike trains at term. To examine mechanisms underlying the plateau, we studied the effects of potassium channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on membrane potentials in CM from rats on gestation Days 14, 15, 16, 21 (term). Apparent membrane conductance was measured at rest and during the plateau in Day 14 muscles with and without TEA. 4-AP depolarized the resting membrane on all gestation days. Therefore, a direct action of 4-AP on plateau configuration could not be separated from an indirect effect of depolarization. TEA did not affect the resting potential but increased action potential size and depolarization rate on all gestation days. On Day 16, TEA reduced plateau amplitude, unmasking small, repetitive depolarizations. D-600 decreased plateau amplitude and duration and attenuated these effects of TEA. Plateau conductance increased initially then decreased before membrane repolarization. Membrane conductance and outward rectification during the plateau were reduced by TEA. The plateau potential may result from an outwardly rectifying TEA-sensitive current combined with a slow inward current, the plateau magnitude being determined by the relative intensity of each current.     

A new scorpion toxin with a very high affinity for sodium channels. An electrophysiological study

The neurotoxic action of toxin gamma from the venom of the Brazilian scorpion Tityus serrulatus (TiTx gamma) has been investigated in cultured mouse neuroblastoma cells (N1E115) using the suction pipette technique. Addition of 14 to 53 nM TiTx gamma to the external solution causes nerve cell membrane depolarization, membrane potential oscillations and spontaneous action potentials within 10 min. None of these effects were observed within 15 min after application of 1 microM toxin IV from Centruroides sculpturatus venom. Under voltage clamp the amplitude of the sodium current evoked by test pulses to potentials more positive than -30 mV is reversibly reduced by 50% after 17 to 105 nM TiTx gamma. On the other hand, a sodium current component appears after TiTx gamma at test pulse potentials between -70 and -40 mV, for which no sodium current is observed in the control experiment. The outward potassium current is not significantly affected by the highest TiTx gamma concentrations used. The potential-dependence of inactivation of the sodium current component that is induced by TiTx gamma is shifted by -30 mV with respect to control values. The local anaesthetic procain at 1 mM discriminates between the two populations of sodium channels observed in the presence of TiTx gamma.     

Effects of physostigmine on the voltage dependent ionic conductances of skeletal muscle fibres

The voltage dependent ionic conductances were studied by analysing the phase plane trajectories of action potentials evoked by electrical stimulation of the sartorius muscles of the frog (Rana esculenta). The delayed outward potassium current was measured also under voltage clamp conditions on muscle fibres of either the frog (Rana esculenta) or Xenopus laevis. On analysing the effect of physostigmine decreasing the peak amplitude, the rate of both the rising and falling phases of the action potentials, it was revealed that the alkaloid at a concentration of 1 mmol/l reduced significantly both the delayed potassium conductance and the outward ionic current values during the action potentials. The inhibition of sodium conductance and inward ionic current was less expressed. The maximum value of delayed potassium conductance measured under voltage clamp conditions was decreased by 1 mmol/l physostigmine. The time constant determined from the development of delayed potassium conductance was increased at a given membrane potential. The voltage vs. n relationship describing the membrane potential dependence of the delayed rectifier was not influenced by physostigmine. It has been concluded that physostigmine changes the time course of the action potentials by decreasing the value of both voltage dependent ionic conductances and by slowing down their kinetics. It is discussed that results obtained from the phase plane analysis of complex pharmacological effects can only be accepted with some restrictions.     

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

Summary The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (P _o ) increased with depolarization;P _o was 0.5 at –40 to –50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1nm at –45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in theP _o vs. V _m relation such that the channels were more likely to be open at potentials <40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.     

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.     

4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers

We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the action potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCl of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 mV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, IK1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

Binding of scorpion toxin to receptor sites associated with sodium channels in frog muscle. Correlation of voltage-dependent binding with activation.

Purified scorpion toxin (Leiurus quinquestriatus) slows inactivation of sodium channels in frog muscle at concentrations in the range of 17-170 nM. Mono[125I]iodo scorpion toxin binds to a single class of sites in frog sartorius muscle with a dissociation constant of 14 nM and a binding capacity of 13 fmol/mg wet weight. Specific binding is inhibited more than 90% by 3 microM sea anemone toxin II and by depolarization with 165 mM K+. Half-maximal inhibition of binding is observed on depolarization to -41 mV. The voltage dependence of scorpion toxin binding is correlated with the voltage dependence of activation of sodium channels. Removal of calcium from the bathing medium shifts both activation and inhibition of scorpion toxin binding to more negative membrane potentials. The results are considered in terms of the hypothesis that activation of sodium channels causes a conformational change in the scorpion toxin receptor site resulting in reduced affinity for scorpion toxin.     

Molecular cloning, genomic organization and functional characterization of a new short-chain potassium channel toxin-like peptide BmTxKS4 from Buthus martensii Karsch(BmK)

Scorpion venom contains many small polypeptide toxins, which can modulate Na(+), K(+), Cl(-), and Ca(2+) ion-channel conductance in the cell membrane. A full-length cDNA sequence encoding a novel type of K(+)-channel toxin (named BmTxKS4) was first isolated and identified from a venom gland cDNA library of Buthus martensii Karsch (BmK). The encoded precursor contains 78 amino acid residues including a putative signal peptide of 21 residues, propeptide of 11 residues, and a mature peptide of 43 residues with three disulfide bridges. BmTxKS4 shares the identical organization of disulfide bridges with all the other short-chain K(+)-channel scorpion toxins. By PCR amplification of the genomic region encoding BmTxKS4, it was shown that BmTxKS4 composed of two exons is disrupted by an intron of 87 bp inserted between the first and the second codes of Phe (F) in the encoding signal peptide region, which is completely identical with that of the characterized scorpion K(+)-channel ligands in the size, position, consensus junctions, putative branch point, and A+T content. The GST-BmTxKS4 fusion protein was successfully expressed in BL21 (DE3) and purified with affinity chromatography. About 2.5 mg purified recombinant BmTxKS4 (rBmTxKS4) protein was obtained by treating GST-BmTxKS4 with enterokinase and sephadex chromatography from 1 L bacterial culture. The electrophysiological activity of 1.0 microM rBmTxKS4 was measured and compared by whole cell patch-clamp technique. The results indicated that rBmTxKS4 reversibly inhibited the transient outward K(+) current (I(to)), delayed inward rectifier K(+) current (I(k1)), and prolonged the action potential duration of ventricular myocyte, but it has no effect on the action potential amplitude. Taken together, BmTxKS4 is a novel subfamily member of short-strain K(+)-channel scorpion toxin.