Biomedical subjects

L D Possani

Publications and source records attributed to L D Possani.

At least 55 records · Page 3Linked to original sources

PubMed1998

Site directed mutants of Noxiustoxin reveal specific interactions with potassium channels.

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N-terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37-39 (Tyr-Asn-Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I-NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.

Animals↗

PubMed1998

Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium-channel-blocking scorpion toxins.

Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.

Amino Acid Sequence↗

PubMed1998

Pandinus imperator scorpion venom blocks voltage-gated K+ channels in human lymphocytes.

Using the patch-clamp technique, we determined that Pandinus imperator scorpion venom blocked whole-cell n-type K+ currents in human peripheral blood lymphocytes in a dose-dependent manner with Kd = 0.02 microgram/ml. K+ channel block was instantaneous and removable by washing with venom-free extracellular solution. The venom-induced block was independent of membrane potential. The venom did not influence activation and inactivation kinetics of the K+ channels, however, accelerated recovery from inactivation. Purified peptides Pi1, Pi2, and Pi3 from the P. imperator venom powerfully blocked Kv1.3 channels in human lymphocytes with Kd values of 9.7 nM, 50 pM, and 0.5 nM, respectively. Flow cytometric membrane potential measurements with the oxonol dye showed that Pi2, the most effective peptide toxin of the P. imperator venom, depolarizes human lymphocytes in accordance with its K+ channel blocking effect.

Barbiturates↗

PubMed1998

A novel toxin form the scorpion Androctonus australis blocks Shaker K+ channels expressed in Xenopus oocytes.

The Shaker B potassium channel expressed in Xenopus laevis oocytes is blocked, in a total reversible manner from the outside part, by a new toxin (Aa1) composed of 40 amino acid residues, purified from the venom of the North African scorpion Androctonus australis Garzoni. The experiments were performed with patch-clamp technique in the outside-out configuration. The half blocking concentration is approximately 4.5 microM with a 1:1 stoichiometry. The activation and inactivation kinetics of the current are not modified by the blocking mechanism. The binding affinity is not voltage dependent. These results suggest a simple bimolecular mechanism of blockade by which the toxin occludes the external vestibule of the channel and thereby inhibits the K+ ions conduction.

Animals↗

PubMed1998

The Androctonus australis garzoni scorpion venom contains toxins that selectively affect voltage-dependent K(+)-channels in cerebellum granular cells.

A purified peptide from Androctonus australis Garzoni venom (AaG) affects selectively a K(+)-current recorded from cerebellum granular cells. This current is characterized by fast activating and inactivating kinetics similar to an IA-type current. Addition of 2 microM peptide Aa1 (from Androctonus australis, toxin 1) to the external side of the channel suppressed completely and in a selective manner the IA-type current, with an IC50 value of 130 nM, whereas in the same conditions, the other potassium current, identified as delayed rectifier (Id), was not affected. Additionally, we show that another partially purified peptide (III-12) from the same venom was able to block reversibly both K(+)-currents.

Animals↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

1H NMR structural analysis of novel potassium blocking toxins using a nano-NMR probe.

A new class of toxin acting on potassium channels and cross-linked by four disulfide bridges instead of three has been recently described. Two peptides, Pi1 and Pi7, purified from the venom of the scorpion Pandinus imperator belong to this new class. Structural features of one of these new toxins. Pi1, have been investigated by proton nuclear magnetic resonance using a new technology that allows to work with very small amount of compound, in the nanomole range. It is shown that it is possible to collect high quality data set in terms of resolution, lineshape and sensitivity with nanomolar amount of compound using this technology. Preliminary results on Pi7 are also presented. The approach described here is quite attractive for the study of natural compounds such as toxins often available at low amounts.

Amino Acid Sequence↗

PubMed1998

From noxiustoxin to Shiva-3, a peptide toxic to the sporogonic development of Plasmodium berghei.

This communication reviews shortly the main structural and functional characteristics of Noxiustoxin, a 39 amino acid residue peptide, maintained closely packed by three-disulfide bridges and its effects on excitable membranes. Shiva-3, a cecropin like-peptide composed of 38 amino acid residues is also briefly reviewed. Its design and synthesis was made possible by the expertise gained through the work previously performed with Noxiustoxin. One of the most prominent functional characteristics of Shiva-3 is the toxic effect upon the sporogonic development of Plasmodium berghei (responsible for a murine version of malaria). A synthetic Shiva-3 gene was constructed by recursive polymerase-chain reaction (PCR) methodology and expressed using the vector pGEX2T as a hybrid protein between the glutathione-S-transferase at the N-terminal and Shiva-3 in the C-terminal part of the hybrid. The recombinant protein kills bacteria and Plasmodium berghei. The future aim of this work is to produce a transgenic mosquito that carries the message for synthesis and excretion of Shiva-3 and similar peptides, in the midgut of mosquitoes, in an attempt to control the spreading of human malaria.

Amino Acid Sequence↗

PubMed1997

The mechanism of inhibition of ryanodine receptor channels by imperatoxin I, a heterodimeric protein from the scorpion Pandinus imperator.

We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.

Amino Acid Sequence↗

PubMed1997

Primary structure and synthesis of Imperatoxin A (IpTx(a)), a peptide activator of Ca2+ release channels/ryanodine receptors.

We present the complete amino acid sequence of Imperatoxin A (IpTx(a)), a 33-amino-acid peptide from the venom of the scorpion P. imperator which activates Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). The amino acid sequence of IpTx(a) shows no homology to any scorpion toxin so far described, but shares some homology to the amino acid sequence of Tx2-9 and agelenin, two spider toxins that target neuronal P-type Ca2+ channels. We also describe the total synthesis of IpTx(a) and demonstrate that it efficiently activates RyRs with potency and affinity identical to those of native IpTx(a). The use of synthetic IpTx(a) should help in the identification of the structural motifs of RyR critical for channel gating.

Amino Acid Sequence↗

PubMed1997

A novel potassium channel blocking toxin from the scorpion Pandinus imperator: A 1H NMR analysis using a nano-NMR probe.

The three-dimensional solution structure of a novel peptide, Pi 1, purified from the venom of the scorpion Pandinus imperator and specific for potassium channels was determined by homonuclear proton NMR methods at 500 MHz from nanomole amounts of compound. P. imperator toxin is a voltage-dependent potassium channel specific peptide capable of blocking the shaker B K+ channels expressed in Sf9 cells in culture (Spodoptera frugiperda cell line no. 9) and displacing labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has only 35 amino acid residues but is stabilized by four disulfide bridges (Cys4-Cys25, Cys10-Cys30, Cys14-Cys32, and Cys20-Cys35) instead of three commonly found in small potassium channel toxins. A detailed nuclear magnetic resonance structure of this protein was obtained using a nano-NMR probe and a combination of two-dimensional proton NMR experiments. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the solution conformation of the toxin. The structure is organized around a short alpha-helix spanning residues Ser8-Thr18 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys10-Cys30 and Cys14-Cys32. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys32 with a tight turn at Arg28-Met29 in contact with the N-terminal fragment Leu1-Cys4. Comparison between the 3D structure of Pi 1 and those of other structurally and functionally related scorpion toxins is presented.

Amino Acid Sequence↗

PubMed1997

Block of ShakerB K+ channels by Pi1, a novel class of scorpion toxin.

Here we describe the basic features of the interaction of K+ channels with Pi1, a recently described 35 amino acid scorpion toxin, which has four disulfide bridges instead of the three commonly found in all the other known scorpion toxins. We found that: (a) Pi1 blocks ShakerB from the outside with a 1:1 stoichiometry, and a Kd of 32 nM in zero external [K+]; (b) extracellular K+, Rb+ and Cs+ but not NH4+ ions strongly impede (destabilize) the block by this toxin; interestingly (c) the destabilizing binding of K+, Rb+, and Cs+ is described by a Hill coefficient n > 1; (d) external K+ is more effective than internal K+ to reduce the block by Pi1.

Animals↗

PubMed1997

Toxins and genes isolated from scorpions of the genus Tityus.

Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.

Amino Acid Sequence↗

PubMed1997

Action of babycurus-toxin 1 from the east African scorpion Babycurus centrurimorphus on the isolated cockroach giant axon.

A toxin named babycurus-toxin 1 (mol. wt 8191), from telson extracts of the scorpion Babycurus centrurimorphus, was found to depolarize the cockroach giant axon. It progressively blocked the evoked action potentials after a short period of limited repetitive activity and after 30 min of toxin action it became impossible to evoke responses to current stimulations. Voltage-clamp experiments on the sodium current indicated that the toxin in micromolar concentrations progressively decreased the transient inward peak sodium current, but also slowed the activation phase of this sodium current and maintained an inward current during the voltage pulses, which deactivated slowly. The toxin also induced in the insect axon a slowly activating-deactivating component of the sodium current. This suggests that the toxin modifies both activation and inactivation mechanisms of sodium channels. Thus there is some similarity in the electrophysiological effects between BcTx1 and the beta-toxins active on mammals.

Amino Acid Sequence↗

PubMed1997

Isolation, characterization and comparison of a novel crustacean toxin with a mammalian toxin from the venom of the scorpion Centruroides noxius Hoffmann.

A novel crustacean-specific toxin, Cn5, containing 66 amino acid residues was isolated from the venom of the scorpion Centruroides noxius Hoffmann. It is stabilized by four disulfide bridges, formed between Cys12-Cys65, Cys16-Cys41, Cys25-Cys46 and Cys29-Cys48. Toxicity tests revealed that Cn5 is a toxin that affects arthropods but not mammals. However, at high concentrations, Cn5 does displace the mammal-specific toxin Cn2 from rat brain synaptosomes. The concentration of Cn5 that produces half-maximal inhibition (IC50) was estimated to be 100 microM. Sequence comparison of Cn5 with toxin Cn2, a mammal-specific toxin from the same scorpion, showed the presence of two sequence stretches, at positions 30 to 38 and 49 to 58, where the majority of the differences are concentrated. On the three-dimensional structure of Cn5 it is demonstrated that these two sequence stretches form a continuous surface region near the site thought to bind to the sodium channel. We assume that this region might be implicated in determining species specificity.

Amino Acid Sequence↗

PubMed1996

An insect-specific toxin from Centruroides noxius Hoffmann. cDNA, primary structure, three-dimensional model and electrostatic surface potentials in comparison with other toxin variants.

Scorpion toxins acting on sodium channels differ in their specificity. Toxic peptides specific towards mammals and arthropods (insects and/or crustaceans) have been described. Because of the similar three-dimensional fold of these peptides, the molecular base of their specificity is thought to reside in certain differences at the level of amino acid residues especially within or near the binding site of the toxin to the particular ion channel. The cDNA, amino acid sequence and biological activity of an insect-specific toxin, Cn10, from the scorpion Centruroides noxius Hoffmann is reported. The electrostatic potential surface around a three-dimensional model of Cn10 was calculated. It revealed that residues Tyr4, Lys13, Ile18, Leu19, Gly20, Lys43, Leu44, Thr57, Tyr58, Pro59, Thr64 and Cys65, situated at the side of the toxin proposed in the literature to bind to the sodium channel, constitute a positive surface region. Therefore, they may form the site that binds to the channel. Cn10 was included in a comparative analysis of two groups of natural variants, highly similar peptides of the genus Centruroides with specificities towards mammals or arthropods. A number of surface-accessible residues, consistently different between the two groups and situated near the putative binding site, may be of importance for the specificity of the analyzed toxins.

Amino Acid Sequence↗

PubMed1996

Two novel alpha-neurotoxins isolated from the taipan snake, Oxyuranus scutellatus, exhibit reduced affinity for nicotinic acetylcholine receptors in brain and skeletal muscle.

Three novel toxic peptides were purified to homogeneity from the venom of the Australian taipan snake, Oxyuranus scutellatus scutellatus. On the basis of complete amino acid sequence analyses, two of these toxins belong to the family of short-chain alpha-neurotoxins found in elapid and hydrophid snake venoms and are the first postsynaptic neurotoxins identified in taipan venom. Radioligand binding studies confirm that taipan toxins 1 and 2 inhibit the binding of [125I]-alpha-bungarotoxin to nicotinic acetylcholine receptors in skeletal muscle with IC50 values of 2.4-2.5 nM but are 5-fold less potent in this assay than alpha-bungarotoxin or the two short-chain alpha-neurotoxins erabutoxin a and erabutoxin b. Taipan toxins 1 and 2 do not antagonize [125I]-alpha-bungarotoxin binding to central neuronal nicotinic receptors at concentrations up to 3 microM. We find that erabutoxin a and erabutoxin b do inhibit the binding of [125I]-alpha-bungarotoxin to central neuronal nicotinic receptors but are over 350-fold less potent than long-chain alpha-neurotoxins at these receptors. The novel alpha-neurotoxins from taipan venom do not inhibit the binding of [3H]nicotine to high-affinity nicotine receptors in brain, a property they share with alpha-bungarotoxin and the erabutoxins. The results demonstrate that at least two neuromuscular junction-blocking peptides are present in taipan venom. Nonconservative substitutions at position 32 in both taipan toxin 1 and 2 may be responsible for the observed decreases in affinities of the toxins of 5-fold for muscle receptors (compared to alpha-bungarotoxin) and over 10-fold for alpha-bungarotoxin-sensitive nicotinic receptors in brain (compared to the structurally similar short-chain alpha-neurotoxins erabutoxin a and erabutoxin b).

Amino Acid Sequence↗

PubMed1996

A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator.

A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4-Cys-25, Cys-10-Cys-30, Cys-14-Cys-32 and Cys-20-Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pil) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.

Amino Acid Sequence↗