Scorpion Buthus Eupeus Research Articles

Species Diversity and Peptide Toxins Blocking Selectivity of Ether-à-go-go-Related Gene Subfamily K+ Channels in the Central Nervous System

The ether-à-go-go-related gene (erg) K+ channels are known to be crucial for life in Caenorhabditis elegans (mating), Drosophila melanogaster (seizure), and humans (LQT syndrome). The erg genes known to date (erg1, erg2, and erg3) are highly expressed in various areas of the rat and mouse central nervous system (CNS), and ERG channel blockers alter firing accommodation. To assign physiological roles to each isoform, it is necessary to design pharmacological strategies to distinguish individual currents. To this purpose, we have investigated the blocking properties of specific peptide inhibitors of hERG1 channels on the human and rat isoforms. In particular, we have tested ErgTx1 (from the scorpion Centruroides noxious), BeKm-1 (from the scorpion Buthus eupeus), and APETx1 (from the sea anemone Anthopleura elegantissima). Because these peptides had different species-specific effects on the six different channels, we have also carried out a biophysical characterization of hERG2 and hERG3 channels that turned out to be different from the rat homologs. It emerged that APETx1 is exquisitely selective for ERG1 and does not compete with the other two toxins. BeKm-1 discriminates well among the three rat members. ErgTx1 is unable to block hERG2, but blocks rERG2 and has the lowest KD for hERG3. BeKm-1 and ErgTx1 compete for hERG3 but not for rERG2 blockade. Our findings should be helpful for structure-function studies and for novel CNS ERG-specific drug design.

An ERG Channel Inhibitor from the Scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.

M-type K + current inhibition by a toxin from the scorpion Buthus eupeus

A number of invertebrate venoms have been tested for effects on M-type K + currents ( I K( M) ) in differentiated mouse neuroblastoma X rat glioma NG108-15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited I K( M) by ∼ 44% at 50 μg/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrymidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited I K( M) by ∼45% with an IC 50 of ∼;33 nM. Neither the crude venom nor the purified polypeptide affected the Ca 2+ current or the delayed rectifier K + current. While the crude venom prolonged the Na + current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of I K( M) in NG108-15 cells.

Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors

We have explored the possibility of improving baculovirus pesticides by incorporating an insect-specific neurotoxin gene into a baculovirus genome. A 112-bp gene ( BeIt) encoding insectotoxin-1 of the scorpion Buthus eupeus was synthesized and cloned in Escherichia coli. For expression, Belt was transferred to the DNA genome of Autographa californica nuclear polyhedrosis virus ( AcMNPV). Three different recombinant AcMNPVs, carrying Belt under the control of the strong AcMNPV polyhedrin promoter, were constructed and expression of Belt was monitored upon infection of Spodoptera frugiperda (Sf) cells. Toxin expression was low using a recombinant virus in which Belt was inserted 6 nucleotides (nt) downstream from the intact polyhedrin mRNA leader. More expression was observed when a signal-peptide was attached in-frame to the N terminus of Belt. The highest level of expression was observed with a fusion gene comprised of the 58 N-terminal codons of polyhedrin fused to Belt; however, the level of expression was ten- to twenty-fold below that for polyhedrin. Polyhedrin promoter-directed transcripts of all three recombinants accumulated to levels similar to those of wild-type polyhedrin transcripts, indicating that the limitation to expression of unfused BeIt was not at the level of transcription but rather at the posttranscriptional level including translation or protein stability. Paralytic activity of the toxin products was not detected.

Solution spatial structure of 'long' neurotoxin M9 from the scorpion Buthus eupeus by 1H-NMR spectroscopy.

1H-NMR spectra of Buthus eupeus neurotoxin M9 (66 amino acid residues, four disulfide bonds) reveal two slowly exchangeable conformations at acidic pH. The spatial structure of the conformer prevailing under physiologically relevant conditions has been determined from two-dimensional 1H-NMR data treated by means of a distance geometry algorithm and refined by molecular modelling. Interrelation between the structure and function of mammalian neurotoxin M9 is discussed by comparing its conformation with those of the scorpion insectotoxins which exhibit different biological specificity (insectotoxins v-2, v-3 and I5A).

Photoactivable neurotoxins in studying membrane receptors

Seven monomodified photoactivable derivatives of neurotoxin M 10 from the scorpion Buthus eupeus venom and neurotoxin II from the cobra Naja naja oxiana venom have been obtained and characterized. By means of these toxin analogs fast sodium channels and the acetylcholine receptor have been studied.

Effects of scorpion (Buthus eupeus) toxins on cyclic AMP and cyclic GMP levels in brain and heart tissues

Toxic polypeptides isolated from venom of the scorpion Buthus eupeus [i, 2] interact selectively with the gating mechanism of the sodium channels of excitable membranes, slowing their inactivation [4]. This action of the toxins on the organ or organism leads to massive liberation of neuromediators from nerve endings [5, 6, 12]. Modification of excitable membranes by certain toxins (anemone toxin ATCP, MCD-peptide from bee venom) is known to stimulate cyclic nucleotide formation [8, 9].

Potential-dependent interaction of toxin from venom of the scorpion Buthus eupeus with sodium channels in myelinated fibre. Voltage clamp experiments

1. 1. The steady-state characteristics of the sodium channel gating in the nodal membrane were determined under voltage clamp conditions before and after treatment with toxins from the venom of scorpion, Buthus eupeus. 2. 2. The apparent binding constant ( K A ) of the toxin was determined for different levels of the membrane potential. At potentials more negative than −120 mV, K A tends to a constant level. K A is maximum at about −80 mV, and it decreases as the potential is reduced to 0 mV. 3. 3. A model assuming that the voltage dependency of K A is mainly due to the difference in electrical energy between inactivated states of normal and poisoned channels is proposed. An additional decrease in overall binding of toxin results from the transition of a fraction of the sodium channels into the state of slow inactivation.

Isolation and characterization of toxins from the venom of Middle-Asian scorpion Buthus eupeus: E. V. Grishin, N. M. Soldatov, L. N. Soldatova and Yu. A. Ovchinnikov Shemyakin Institute of Bioorganik Chemistry, USSR Academy of Sciences, Moscow, U.S.S.R.

Isolation and characterization of toxins from the venom of Middle-Asian scorpion Buthus eupeus: E. V. Grishin, N. M. Soldatov, L. N. Soldatova and Yu. A. Ovchinnikov Shemyakin Institute of Bioorganik Chemistry, USSR Academy of Sciences, Moscow, U.S.S.R.

Effects of scorpion venom on squid axon membranes.

Effects of scorpion venom on squid axon membranes.