Froylan Gómez-Lagunas

Oxyopinins, Large Amphipathic Peptides Isolated from the Venom of the Wolf Spider Oxyopes kitabensis with Cytolytic Properties and Positive Insecticidal Cooperativity with Spider Neurotoxins

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially α-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.

Two novel toxins from the Amazonian scorpion Tityus cambridgei that block Kv1.3 and Shaker B K+-channels with distinctly different affinities

Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.

SHAKER B K+ CONDUCTANCE IN NA+ SOLUTIONS LACKING K+ IONS : A REMARKABLY STABLE NON-CONDUCTING STATE PRODUCED BY MEMBRANE DEPOLARIZATIONS

1. Shaker B K+ channels, expressed in the insect cell line Sf9, were studied in zero K+, Na+ or N‐methyl‐D‐glucamine (NMG)‐containing solutions. In the absence of K+ ions on both sides of the membrane, the K+ conductance collapsed with the delivery of short depolarizing pulses that activated the channels. The collapse of the conductance was fully prevented when the channels were kept closed at a holding potential of ‐80 mV. 2. The fall in K+ conductance had the notable characteristic of being strikingly stable. At ‐80 mV or more negative holding potentials, the conductance never recovered (cells observed for up to 1 h). 3. The extent of collapse of the K+ conductance depended on the number of depolarizing activating pulses applied in zero K+ solutions. For moderate to low frequencies of pulsing (1 to 0.002 Hz), the extent of the collapse did not depend on the frequency. 4. K+, Rb+, Cs+ and NH4+ added to the external Na+ solution impeded the fall in K+ conductance. 5. TEA added to the external, zero K+, Na(+)‐containing solution also precluded the fall of the conductance. The protection by TEA paralleled its block of the outward K+ currents recorded with standard recording solutions. 6. The fall in K+ conductance was prevented by depolarized holding potentials. 7. The K+ conductance that was thought to be irreversibly lost at ‐80 mV or more negative holding potentials was fully recovered, however, after a prolonged (tens of seconds to minutes) change in the holding potential to depolarized values (above ‐50 mV). Full recovery could be obtained at any time after the former halt of the conductance.

Conserved motifs in mechanosensitive channels MscL and MscS

Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea.

Tc1, from Tityus cambridgei, is the first member of a new subfamily of scorpion toxin that blocks K+-channels

A new peptide, Tc1, containing only 23 amino acids closely packed by three disulfide bridges was isolated from the Amazonian scorpion Tityus cambridgei. It blocks reversibly the Shaker B K+‐channels with a K d of 65 nM and displaces binding of noxiustoxin to mouse brain synaptosome membranes. It is the shortest known peptide from scorpion venom that recognizes K+‐channels and constitutes a new structural subfamily of toxin, classified as alphaKTx 13.1.

The relation between ion permeation and recovery from inactivation of ShakerB K+ channels.

We have studied the relation between permeation and recovery from N-type or ball-and-chain inactivation of ShakerB K channels. The channels were expressed in the insect cell line Sf9, by infection with a recombinant baculovirus, and studied under whole cell patch clamp. Recovery from inactivation occurs in two phases. The faster of the two lasts for approximately 200 ms and is followed by a slow phase that may require seconds for completion. The fast phase is enhanced by both permeant ions (K+, Rb+) and by the blocking ion Cs+, whereas the impermeant ions (Na+, Tris+, choline+) are ineffective. The relative potencies are K+ > Rb+ > Cs+ > NH4+ >> Na+ approximately choline+ approximately Tris+. Ion permeation through the channels is not essential for recovery. The results suggest that cations influence the fast phase of recovery by binding in a site with an electrical distance greater than 0.5. Recovery from fast inactivation is voltage-dependent. With Na+, choline+, or Tris+ outside, about 15% of the channels recover in the fast phase (-80 mV), and the other 85% apparently enter a second inactivated state from which recovery is very slow. Recovery in this phase is not influenced by external ions, but is speeded by hyperpolarization.

Inactivation in ShakerB K+ channels: a test for the number of inactivating particles on each channel

Fast inactivation in ShakerB K channels results from pore-block caused by ball peptides attached to the inner part of each K channel. We have examined the question of how many functional inactivating balls are on each channel and how this number affects inactivation and recovery from inactivation. To that purpose we expressed ShakerB in the insect cell line Sf9 and gradually removed inactivation by perfusing the cell interior with the hydrolytic enzyme papain under whole cell patch clamp. Inactivation slows down as the balls are removed by an amount consistent with the presence of four balls on each channel. Recovery from inactivation has the same time course early and late in papain action; it does not depend on the number of balls remaining on the channel, consistent with the idea that reinactivation is not significant during recovery from inactivation. Our conclusion is that ShakerB has four ball peptides, each capable of causing inactivation. Statistically, the balls are identical and independent. The stability of N-type inactivation by the remaining balls is not appreciably affected by removing some of the balls from a channel.

Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels

Abstract. Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K+ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K+ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K+ channels with a Kd of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of 125I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.

Two similar peptides from the venom of the scorpion Pandinus imperator, one highly effective blocker and the other inactive on K+ channels

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K+-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K+ -channels (a Kv1.1 sub-family type of channel) at 100nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K+-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with Ic50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important point mutations responsible for such impressive variation in blocking properties of both toxins, here described.

Disulfide bridges and blockage of Shaker B K(+)-channels by another butantoxin peptide purified from the Argentinean scorpion Tityus trivittatus.

A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.