Paulo S.L. Beirão

Functional and structural features of γ-zeathionins, a new class of sodium channel blockers

γ1‐ and γ2‐zeathionins (γ1‐Z and γ2‐Z) are members of a family of small and basic peptides involved in plant protection. These plant defensins exhibit remarkable structural similarity to scorpion neurotoxins and insect defensins. In the present report, we used the whole‐cell patch clamp technique to investigate the inhibition of the sodium current (I Na) by γ1‐Z and γ2‐Z in the GH3 cell line. Both γ1‐Z and γ2‐Z rapidly and reversibly inhibited I Na without changing the kinetics or voltage dependence of activation or inactivation. To our knowledge, this is the first example of a plant protein that inhibits the sodium channel. From structural comparisons with the μ‐conotoxins, a family of peptides that block the sodium channel, we detected some similar features that could provide the basis of inhibition of sodium channels by γ‐zeathionins.

α-Scorpion Toxin Impairs a Conformational Change that Leads to Fast Inactivation of Muscle Sodium Channels

α-Scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an α-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence–voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

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.

β-Scorpion Toxin Modifies Gating Transitions in All Four Voltage Sensors of the Sodium Channel

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

Inhibition of neuronal high-voltage activated calcium channels by the ω-Phoneutria nigriventer Tx3-3 peptide toxin

We have investigated the effect of omega-PnTx3-3 (referred to in previous papers simply as Tx3-3), a peptide toxin from the venom of the spider Phoneutria nigriventer, on neuronal high-voltage activated (HVA) Ca(2+) channels, using whole-cell patch-clamp. omega-PnTx3-3 (120 nM) blocked 74+/-8% of the total HVA Ca(2+) currents of cerebellar granule neurones, without affecting the low-voltage activated (LVA) current. P/Q/R-type currents in cerebellar granule neurones, isolated using 4 microM nicardipine and 100 nM omega-conotoxin GVIA, were markedly (79+/-6%) inhibited by 60 nM omega-PnTx3-3. R-type currents, isolated either by additional application of 0.5-1 microM of omega-agatoxin IVA or by pre-incubation with 5 microM omega-conotoxin MVIIC were inhibited almost totally by 120 nM of omega-PnTx3-3. omega-PnTx3-3 reversibly altered the kinetics of the P/Q/R current, increasing the degree of inactivation that occurred during a 50 ms pulse from 20% to 40%. N-type currents, recorded from neuroblastoma N18 cells, were partially (34+/-2%) inhibited by 320 nM omega-PnTx3-3. L-type currents, recorded from GH3 cells, were partially (45+/-12%) inhibited by 80 nM omega-PnTx3-3. We conclude that omega-PnTx3-3 inhibits all known HVA Ca(2+) channels, and most effectively the P/Q- and R-type currents.

Electrophysiological characterization and molecular identification of the Phoneutria nigriventer peptide toxin PnTx2-61

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2‐6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate‐rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2‐6. The purified toxin exerted complex effects on Na+ current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na+ conductance and the steady‐state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K D of 0.8 μM for the toxin–channel complex.

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer).

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.

Cloning of cDNAS encoding neurotoxic peptides from the spider Phoneutria nigriventer.

A cDNA library made from venom glands of the spider Phoneutria nigriventer was constructed and used to clone neurotoxic peptides. A cDNA of about 360 nucleotides encoding the precursor for the toxin Tx2-1 active on mammals has been isolated. The deduced amino acid sequence for the mature polypeptide confirms the polypeptide sequence previously published. In addition, two new putative toxins called Pn2-1A and Pn2-5A have been characterized and their complete amino acid sequence show 92% similarity to Tx2-1 and 94% similarity to Tx2-5 respectively. The cDNAs revealed that the precursors contain signal peptides characterized by a very hydrophobic core and a propeptide interposed between the signal sequence and the peptide toxin.