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.
Two-dimensional 1H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for ...two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and c-termini of each chain) are 0.75 A for backbone heavy atoms, and 1.25 A for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two alpha-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-alpha-helical bundle structure.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of δ-latroinsectotoxin (δ-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. δ-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of δ-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (~130 kDa) encoded in the δ-LIT gene differs from that of native δ-LIT purified from BWSV (~110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native δ-LIT revealed a molecular ion with m/z+ of 110916 ± 100, indicating that the native δ-LIT is 991 amino acids in length. When the full-length δ-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native δ-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.
The venom of the black widow spider (BWSV) ( Latrodectus mactans tredecimguttatus ) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific
manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown,
and they have not been functionally expressed. This study reports on the primary structure of -latroinsectotoxin ( -LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. -LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree
of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain
organization of -LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted
molecular mass and apparent mobility of the protein ( 130 kDa) encoded in the -LIT gene differs from that of native -LIT purified from BWSV ( 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native -LIT revealed a molecular ion with m/z + of 110916 ± 100, indicating that the native -LIT is 991 amino acids in length. When the full-length -LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing
991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar
concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native -LIT have a high Ca permeability, whereas those formed by truncated, recombinant protein do not.
A novel polypeptide, designated omega-Lsp-IA, which modulates P-type Ca(2+) channels, was purified from the venom of the spider Geolycosa sp. omega-Lsp-IA contains 47 amino acid residues and 4 ...intramolecular disulfide bridges. It belongs to a group of spider toxins affecting Ca(2+) channels and presumably forms the inhibitor cystine knot (ICK) fold. Peculiar structural features (a cluster of positively charged residues in the C-terminal loop of the peptide and a regular distribution of hydrophobic residues) that may play a decisive role in the omega-Lsp-IA mechanism of action were located. Recombinant omega-Lsp-IA was produced in prokaryotic expression system and was shown to be structurally and functionally identical to the native toxin. At saturating concentration (10nM), the peptide clearly slows down the activation kinetics and partially inhibits the amplitude of P-current in rat cerebellar Purkinje neurons. Prominent deceleration of the activation kinetics is manifested as the appearance of a five-fold slower component of the current activation. The specificity of action of omega-Lsp-IA on different Ca(2+) channel types was studied in isolated hippocampal neurons of rat. omega-Agatoxin IVA completely removed the effect of omega-Lsp-IA on the whole-cell Ca(2+) current. Therefore, omega-Lsp-IA appears to act specifically on P-type Ca(2+) channels.
The proton NMR spectra at 300 MHz of neurotoxin III from venom of Naja mossambica mossambica are reported. By the use of double resonance techniques, pH dependence chemical shifts, isotope labeling ...technique, and comparison with homologous neurotoxins all proton signals in the aromatic and methyl regions as well as ɛ‐CH2 proton signals of some lysine residues have been assigned to individual amino acid residues and their spatial microenvironment has been determined. The results deduced on the solution structure of neurotoxin III are in complete agreement with the crystal structure of sea snake erabutoxins as well as with the previously established backbone folding and inter‐residue interactions for the Naja naja oxiana short‐chain neurotoxin in solution. In addition evidence has been obtained (a) that the conformation of the β turn in the 31–34 segment depends on the ionization state of the Asp‐31 and His‐32 side chain groups and (b) that an intricate electrostatic interaction exists in a system of ionogenic groups of the invariant Lys‐27, Lys‐47, Asp‐31, Arg‐33, Glu‐38 and His‐32 residues. These aspects of dynamic conformation are related to an interaction mechanism of a neurotoxin molecule and a nicotinic acetylcholine receptor.
We have identified a novel polypeptide toxin (Lsp-1) from the venom of the spider Lycosa (LS). Its effect has been examined on the P-type calcium channels in Purkinje neurons, using whole-cell ...patch-clamp. This toxin (at saturating concentration 7 nM) produces prominent (four-fold) deceleration of the activation kinetics and partial (71+/-6%) decrease of the amplitude of P-current without affecting either deactivation or inactivation kinetics. These effects are not use-dependent. They are partially reversible within a minute upon the wash-out of the toxin. Intracellular perfusion of Purkinje neurons with 100 microM of GDP or 2 microM of GTPgammaS, as well as strong depolarising pre-pulses (+100 mV), do not eliminate the action of Lsp-1 on P-channels indicating that down-modulation via guanine nucleotide-binding proteins (G-proteins) is not involved in the observed phenomenon. In view of extremely high functional significance of P-channels, the toxin can be suggested as a useful pharmacological tool.
A novel polypeptide, designated
ω-Lsp-IA, which modulates P-type Ca
2+ channels, was purified from the venom of the spider
Geolycosa sp.
ω-Lsp-IA contains 47 amino acid residues and 4 intramolecular ...disulfide bridges. It belongs to a group of spider toxins affecting Ca
2+ channels and presumably forms the inhibitor cystine knot (ICK) fold. Peculiar structural features (a cluster of positively charged residues in the C-terminal loop of the peptide and a regular distribution of hydrophobic residues) that may play a decisive role in the
ω-Lsp-IA mechanism of action were located. Recombinant
ω-Lsp-IA was produced in prokaryotic expression system and was shown to be structurally and functionally identical to the native toxin. At saturating concentration (10
nM), the peptide clearly slows down the activation kinetics and partially inhibits the amplitude of P-current in rat cerebellar Purkinje neurons. Prominent deceleration of the activation kinetics is manifested as the appearance of a five-fold slower component of the current activation. The specificity of action of
ω-Lsp-IA on different Ca
2+ channel types was studied in isolated hippocampal neurons of rat.
ω-Agatoxin IVA completely removed the effect of
ω-Lsp-IA on the whole-cell Ca
2+ current. Therefore,
ω-Lsp-IA appears to act specifically on P-type Ca
2+ channels.
We have identified a novel polypeptide toxin (Lsp-1) from the venom of the spider
Lycosa (LS). Its effect has been examined on the P-type calcium channels in Purkinje neurons, using whole-cell ...patch-clamp. This toxin (at saturating concentration 7
nM) produces prominent (four-fold) deceleration of the activation kinetics and partial (71
±
6%) decrease of the amplitude of P-current without affecting either deactivation or inactivation kinetics. These effects are not use-dependent. They are partially reversible within a minute upon the wash-out of the toxin. Intracellular perfusion of Purkinje neurons with 100
μM of GDP or 2
μM of GTPγS, as well as strong depolarising pre-pulses (+100
mV), do not eliminate the action of Lsp-1 on P-channels indicating that down-modulation via guanine nucleotide-binding proteins (G-proteins) is not involved in the observed phenomenon. In view of extremely high functional significance of P-channels, the toxin can be suggested as a useful pharmacological tool.
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