Dean D. Watt

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

Abstract1.The effect of various toxin fractions isolated by Watt et al. (1978) from the venom of the scorpionCentruroides 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 μg/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 theINa (E) curve to more negative potentials. The amount of shift varied between −10 and −60 mV. A 500ms 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 theINa (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 that of toxin V.6.All effects observed with toxin III or IV were also seen with the whole venom (cf. Cahalan 1975).

Amino acid sequences of neurotoxic protein variants from the venom of Centruroides sculpturatus Ewing

Abstract The venom from the scorpion, Centruroides sculpturatus Ewing (range, Southwestern United States), was fractionated into ten protein zones by chromatography on CM-cellulose. Further purification of three of these zones on DEAE Sephadex in ammonium acetate developers yielded three principal neurotoxins designated variants 1, 2, and 3. Variants 1 and 3 each consist of 65 amino acid residues, variant 2 is composed of 66 residues, and each variant is a single polypeptide chain crosslinked by four disulfide bridges. The three variants have lysine at the amino terminus, serine at the carboxyl terminus, and their sequences exhibit a high degree of homology. The complete structure of variant 2 was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. The sequences of most of the tryptic peptides of variants 1 and 3 were determined, and the peptides were aligned by comparison with the homologous peptides in variant 2. The results show that there are 4 differences in sequence between variants 2 and 3 and 9 differences between variants 1 and 2. Variants 1 and 3 differ from each other at 5 positions in their sequences. These differences between the three protein variants are found in the amino terminal one-third of the molecules except for the deletion of one seryl residue at the carboxyl terminal of variants 1 and 3.

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue. The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions. The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.

Interactions of Scorpion Toxins with the Sodium Channel

It is evident from the data reviewed that scorpion toxins can be distinguished on the basis of three properties: their effects on Na currents, their specific binding to excitable membranes, and the effects of depolarization and pH on binding and on effect. Additional work with other scorpion toxins is required to establish the degree of correlation between the three properties for each class of toxin. Further investigations with this family of homologous proteins will undoubtedly contribute not only to our understanding of the toxins themselves but also to our understanding of the structure and function of the Na channel.

Architecture of scorpion neurotoxins: a class of membrane-binding proteins

Abstract The three-dimensional structure of a scorpion neurotoxin has been determined from high-resolution crystallographic data. The protein possesses a large flattened surface that contains many of the conserved residues and a high concentration of hydrophobic residues. It is likely that other scorpion toxins have this same overall structure, and that they bind to excitable membranes through sites on the conserved-hydrophobic surface of the molecule.

Classification of Na channel receptors specific for various scorpion toxins

Abstract1. The specific binding to rat brain synaptosomes of a radiolabelled derivative of toxin II from the scorpionCentruroides suffusus suffusus could be prevented by toxins III and IV, but not by toxin V or variants 1–3, from the venom ofCentruroides sculpturatus.2. The specific binding of a similar derivative of toxin II fromAndroctonus australis Hector was not affected by any of the toxins fromCentruroides sculpturatus.3. There is biochemical evidence for only two distinct classes of Na channel receptors specific for known scorpion toxins.

Solution structure of an Old World-like neurotoxin from the venom of the New World scorpion Centruroides sculpturatus Ewing

We have determined the solution structure of an alpha-toxin, CsE-V, isolated from the venom of the New World scorpion Centruroides sculpturatus Ewing (CsE). This toxin causes spontaneous rhythmic contractions in muscle. Unlike other New World toxins from CsE, this protein exhibits amino acid insertions and deletions at locations similar to Old World toxins and may thus represent a transition protein between the New World and Old World scorpion alpha-toxins. Sequence-specific assignments were made using 600 MHz 1H two-dimensional NMR data. NOESY, PH-COSY and amide-exchange data were used to deduce constraints for molecular modeling calculations. Distance geometry and dynamical simulated annealing calculations were performed to generate a family of 70 structures free of constraint violations. With respect to this family of structures, the energy-minimized average structure had root-mean-square deviations of 0.74 and 1.32 A for backbone and all atoms, respectively (excluding the C-terminal dipeptide, which is disordered). As with other scorpion toxins, the secondary structure of CsE-V consists of an alpha-helix, a three-strand anti-parallel beta-sheet, four beta-turns, and a hydrophobic patch that includes tyrosine residues in herringbone configuration. Unlike the CsE-v3 and -v1 proteins from C. sculpturatus, all of the proline residues were found to be in the trans configuration. The alpha-helix is slightly longer in CsE-V. The overall structure is more similar to the Old World alpha-toxin AaH-II from Androctonus australis Hector (r.m.s.d 1.59 A for backbone atoms of matching residues) than to the New World alpha-toxin CsE-v3 (r.m.s.d. 1.91 A). These structural data on CsE-V add further to our knowledge of the conformational repertoire exhibited by these sodium channel-binding neurotoxins.

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K(+) channels.

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K(+) channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastomaxglioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12microg/ml.

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

Abstract1.In voltage clamped nodes of Ranvier inactivation of the sodium permeability is slowed by toxin V from the scorpionCentruroides 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.2.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.3.To study the phenomenon quantitatively we fitted the decaying phase of the inward Na current with the equationINa=A exp(-t/τ1)+B exp(-t/τ2)+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 τ1 and τ1.4.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 τ and the steady-state value depended on the potential during the conditioning pulse. For nodes treated with toxin V, τ was 0.24 s at 0 mV and 12° C and half inhibition occurred at −42 mV. The time constant τ 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.5.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.6.The results are interpreted by assuming that a sepolarizing 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 quantitative study of the action ofCentruroides sculpturatus toxins III and IV on the Na currents of the node of Ranvier

Abstract1.In nodes of Ranvier treated with toxin III or IV from the scorptionCentruroides sculpturatus Ewing a strong positive pulse is followed by a transient shift of the descending branch of theINa(E) curve to more negative values of membrane potential (cf. Meves et al. 1982). This effect was studied in more detail, using toxin concentrations between 0.8 and 3.3 μg/ml.2.The change of theINa(E) curve was accompanied by a shift of the kinetic parameters of both activation (time to peak, time constant τm) and inactivation (time constant τh1). The τm curve was shifted by the same amount as the descending branch of theINa(E) curve while the shift of τh1 was somewhat smaller. The curve relating Na permeability to membrane potential became less steep and its lower part was shifted to more negative values of membrane potential.3.The change of theINa(E) curve was also accompanied by a change in the turning-on kinetics of the Na current. The normal signoidal time course was replaced by first-order kinetics. A strong hyperpolarizing prepulse restored the sigmoidal time course without abolishing the shift of the descending branch of theINa(E) curve.4.The transient change of theINa(E) curve was not accompanied by a marked change in the ion selectivity of the Na channels: the equilibrium potential decreased only by 4–8 mV.5.Ca slightly reduced the shift of the descending branch of theINa(E) curve. The long-lasting inward Na current which follows a strong positive pulse in nodes treated with toxin III or IV was reduced by 7.8 mM Ca to 41% of the value measured in normal *1.8 mM) Ca. Mg was slightly less effective than Ca.6.From the change of the Na permeability curve the percentage of Na channels transiently modified by a strong positive pulse was estimated as about 50%.