Marta N. Cordeiro

Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria Nigriventer (keys.)

Six neurotoxic peptides (Tx3-1 to Tx3-6) were purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse phase FPLC on PEP-RPC and PRO-RPC columns, reverse phase HPLC on Vydac C18, and ion exchange HPLC on cationic and anionic columns. These toxins caused different neurological symptoms in mice after intracerebroventricular injection. At dose levels of 5 micrograms/mouse, Tx3-3 and Tx3-4 caused rapid general flaccid paralysis followed by death in 10-30 min; Tx3-2 induced immediate clockwise gyration and flaccid paralysis after 6 hr; Tx3-1, Tx3-5 and Tx3-6 produced paralysis only in the posterior limbs and gradual decreases in movement and aggression during 24 hr. The mol. wt of these cystine-rich peptides were found to be in the range of 3500-8500 by mass spectroscopy and SDS-PAGE. The complete amino acid sequences of the neurotoxins Tx3-1 (40 residues), Tx3-2 (34 residues) and Tx3-6 (55 residues), and the N-terminal sequences of Tx3-3 (34 res.), Tx3-4 (40 res.) and Tx3-5 (36 res.) were established by direct automated Edman degradation, and manual DABITC/PITC microsequence analyses of peptides obtained from digests with various proteases. The structures of these Tx3 neurotoxins from Phoneutria nigriventer exhibited sequence similarities to one another and to the neurotoxins from the venoms of the spiders Hololena curta and Agelenopsis aperta, which were most evident in the location of the Cys residues.

The purification and amino acid sequences of four Tx2 neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keys)

Four neurotoxic polypeptides (Tx2.1, Txt2‐5, Tx2‐6 and Tx2‐9) were purified from the venom of the South American ‘armed’ spider Phoneutria nigriventer (Keys) by gel filtration and reverse phase FPLC and HPLC. These cysteine‐rich polypeptides exhibited different levels of neurotoxicity in mice after intracerebroventricular injection. Tx2‐1, Tx2‐5 and Tx2‐6 caused spastic paralysis and death, but the less toxic Tx2‐9 produced only tail erection and scratching. The molecular weights of the polypeptides as determined by desorption mass spectroscoopy were 5838.8 for Tx2‐1, 5116.6 (Tx2‐5), 5291.3 (Tx2‐6) and 3742.1 (Tx2‐9). The complete amino acid sequences of the neurotoxins were determined by automated Edman degradation and by manual DABITC‐PITC microseqeunce analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2‐1 (53 residues), Tx2‐5 (48 residues) and Tx2‐6 (48 residues) were homologous, but had only limited similarities to the less toxic Tx2‐9 (32 residues). All four polypeptides had varying sequence identities with other neurotoxins from different spider species and biologically active peptides from scorpions, a sea snail and seeds of Mirabilis jalapa.

Isolation of neurotoxic peptides from the venom of the ‘armed’ spider Phoneutria nigriventer

Three neurotoxic fractions, lethal to mice, were isolated from the venom of the spider Phoneutria nigriventer, by gel filtration and reverse phase chromatography (Phoneutria toxins 1, 2 and 3). These toxins have mol. wts in the range 6000-9000, and have different amino acid compositions and N-terminal amino acid sequences. The toxins also differ in the lethality and signs they cause in mice after intracerebro-ventricular injection. The median LD50 being respectively for the whole venom, toxins 1, 2 and 3, 47 +/- 5 micrograms, 45 +/- 4 micrograms, 1.7 +/- 0.7 micrograms and 137 +/- 10 micrograms/kg mouse. Toxins 1 and 2 induce excitatory symptoms in mice and toxin 3 a flaccid paralysis with an ED50 of 40 +/- 5 micrograms/kg mouse as measured also by intracerebro-ventricular injection. The presence in the venom of a non-neurotoxic, smooth muscle active peptide is also described.

Effects of a toxic fraction, PhTx2, from the spider Phoneutria nigriventer on the sodium current.

SummaryThe toxic fraction PhTx2 of the spider Phoneutria nigriventer was studied with a modified loose patch clamp technique on frog skeletal muscle. At saturating concentration (8 μg/ml) potassium currents were unaffected whereas there was a 7-fold increase in the time constant of sodium current inactivation (at −13 mV test potential). The time course of tail current deactivation was at least 3-fold slower than the control. The steady state (100 ms) inactivation and the conductance activation were shifted toward more negative potentials by 12.2 and 7.0 mV, respectively. The reversal of the sodium current was shifted 7.6 mV to more negative potential. We conclude that PhTx2 prolongs the inactivation and deactivation processes of sodium ion channels. These effects may account for the toxicity of PhTx2.

Phoneutria nigriventer toxin Tx3-1 blocks A-type K+ currents controlling Ca2+ oscillation frequency in GH3 cells.

Abstract: GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3‐1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3‐1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4‐aminopyridine and charybdotoxin. Whole‐cell patch clamp experiments show that Tx3‐1 reversibly inhibits the A‐type K+ current (IA) but does not block other K+ currents (delayed‐rectifying, inward‐rectifying, and large‐conductance Ca2+‐sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3‐1, which shows that Tx3‐1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3‐1 is a selective inhibitor of IA, which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3‐1, we suggest that IA is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1 The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3‐3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2 Tx3‐3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3‐3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1‐43. 3 Using ω‐toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3‐3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage‐sensitive calcium channels altered by Tx3‐3 can also be inhibited by ω‐agatoxin IVA, an antagonist of P/Q calcium channels. ω‐conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4 It is concluded that Tx3‐3 potently inhibits ω‐agatoxin IVA‐sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Analgesic effect in rodents of native and recombinant Phα1β toxin, a high-voltage-activated calcium channel blocker isolated from armed spider venom

Abstract Calcium influx through neuronal voltage‐sensitive calcium channels (VSCCS) mediates nociceptive information in the spinal dorsal horn. In fact, spinally administered VSCCS blockers, such as ω‐conotoxin MVIIA, have analgesic effect apart of their low therapeutic index and many side effects. Here we study the analgesic potential of Phα1β, a calcium channel blocker, in rodent models of acute and persistent pain. Spinally administered Phα1β showed higher efficacy and long‐lasting analgesia in a thermal model of pain, when compared with ω‐conotoxin MVIIA. Moreover, Phα1β was more effective and potent than ω‐conotoxin MVIIA not only to prevent, but especially to reverse, previously installed persistent chemical and neuropathic pain. Furthermore, the analgesic action of both toxins are related with the inhibition of Ca2+‐evoked release of pro‐nociceptive neurotransmitter, glutamate, from rat spinal cord synaptosomes and decrease of glutamate overflow in cerebrospinal fluid. When side effects were assessed, we found that Phα1β had a therapeutic index wider than ω‐ conotoxin MVIIA. Finally, recombinant Phα1β expressed in Escherichia coli showed marked analgesic activity similar to the native toxin. Taken together, the present study demonstrates that native and recombinant Phα1β have analgesic effects in rodent models of pain, suggesting that this toxin may have potential to be used as a drug in the control of persistent pathological pain.

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer

A lethal neurotoxic polypeptide of Mr 8kDa was purified from the venom of the South American ‘armed’ or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S‐carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.

Purification and amino acid sequence of a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria nigriventer which inhibits NMDA-evoked currents in rat hippocampal neurones

A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly (Musca domestica), cockroach (Periplaneta americana) and cricket (Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with uncoordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 microg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 microM) reversibly inhibited the N-methyl-D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or gamma-aminobutyric acid-activated currents.