MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABAA receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α(+)/ß(-) interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABAA receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.

Expression of the standard scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements.

The AaH II toxin from the scorpion Androctonus australis Hector is considered to be the standard alpha-toxin because it selectively binds with the highest known affinity to site 3 of mammalian voltage-activated Na+ channels (Na(v)) on rat brain synaptosomes but does not bind to insect synaptosomes. We generated two different constructs in pMALp allowing us to produce AaH II fused with the maltose-binding protein (MBP) in E. coli. We obtained reasonable amounts of recombinant AaH II after cleavage by enterokinase at the site DDDDK. We show that the introduction of a net negative charge at the C-terminus by the suppression of H64 amidation and the addition of an extra residue to the C-terminus (G65) led to fully active AaH II mutants, exhibiting exactly the same affinity as the native toxin for its target on rat brain synaptosomes. In contrast, the mutation of residue K58 into V, I or E residues drastically reduced toxin activity.

Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.

A first exploration of the venom of the Buthus occitanus scorpion found in southern France.

Even though Buthus occitanus scorpions are found throughout the Mediterranean region, a lack of distinctive characteristics has hampered their classification into different subspecies. Yet, stings from this particular scorpion family are reported each year to result in pain followed by various toxic symptoms. In order to determine the toxicity origin of the rare French B. occitanus Amoreux scorpion, we collected several specimens and studied their venom composition using a nano ultra high performance liquid chromatography and matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (nano UHPLC/MALDI-TOF-MS) automated workflow combined with an enzyme-linked immunosorbent assay (ELISA) approach. Moreover, we compared this dataset to that obtained from highly lethal Androctonus australis and Androctonus mauretanicus scorpions collected in North Africa. As a result, we found that the B. occitanus Amoreux venom is toxic to mice, an observation that is most likely caused by venom components that inhibit voltage-gated sodium channel inactivation. Moreover, we identified similarities in venom composition between B. occitanus scorpions living in the South of France and other Buthidae collected in Morocco and Algeria. As such, the results of this study should be taken into consideration when treating stings from the B. occitanus species living in the South of France.

Characterization of the first K⁺ channel blockers from the venom of the Moroccan scorpion Buthus occitanus Paris.

The availability of a large variety of specific blockers, which inhibit different K(+) currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca(2+)-activated K(+) channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using (125)I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the (125)I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family.

Characterization of three "Birtoxin-like" toxins from the Androctonus amoreuxi scorpion venom.

The venom of the North African scorpion Androctonus amoreuxi (Aam) was analyzed using a combination of gel filtration, C18 reverse phase HPLC together with mass spectrometry analysis and bioassays. Three novel Birtoxin-like (BTX-L) peptides of 58 amino acid residues comprising three disulfide bridges were isolated and chemically characterized. One peptide, AamBTX-L3, induced serious toxic symptoms in mice and was lethal at nanogram quantities using intracerebroventricular injection. The three BTX-L peptides were tested in competition experiments on rat brain synaptosomes against the (125)I-labeled "classical" α- and ß-toxins of reference, as well as with the (125)I-KTX, a voltage-gated potassium channel blocker. Only AamBTX-L3 was able to prevent the equilibrium binding of the ß-toxin (125)I-Css IV to its receptor site 4 with a IC(50) value of 189 nM. Even if previous electrophysiological data allowed the classification of other BTX-L peptides among the ß-type toxins, this report clearly shows that AamBTX-L3 is pharmacologically a ß-toxin, which recognizes the voltage-gated Na(+) (Na(v)) channels from central mammalian neurons. In order to uncover the residues functionally essential for interaction between the AamBTX-L3 with the putative receptor site of (125)I-Css IV on Na(v)1.2, molecular models of the three novel Aam BTX-L molecules were made and their surfaces were compared to the already described Css IV biologically interactive surfaces. A hypothesis is given that in BTX-L3, three residues found in the α-helix play a key role during target binding.

Full characterization of three toxins from the Androctonus amoreuxi scorpion venom.

In this study, we have characterized the immunological and pharmacological properties of the three major alpha-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of alpha-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as alpha-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.

A common "hot spot" confers hERG blockade activity to alpha-scorpion toxins affecting K+ channels.

While alpha-KTx peptides are generally known for their modulation of the Shaker-type and the Ca(2+)-activated potassium channels, gamma-KTxs are associated with hERG channels modulation. An exception to the rule is BmTx3 which belongs to subfamily alpha-KTx15 and can block hERG channels. To explain the peculiar behavior of BmTx3, a tentative "hot spot" formed of 2 basic residues (R18 and K19) was suggested but never further studied [Huys I, et al. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K(+) and HERG currents. Biochem J 2004;378:745-52]. In this work, we investigated if the "hot spot" is a commonality in subfamily alpha-KTx15 by testing the effect of (AmmTx3, Aa1, discrepin). Furthermore, single mutations altering the "hot spot" in discrepin, have introduced for the very first time a hERG blocking activity to a previously non-active alpha-KTx. Additionally, we could extend our results to other alpha-KTx subfamily members belonging to alpha-KTx1, 4 and 6, therefore, the "hot spot" represents a common pharmacophore serving as a predictive tool for yet to be discovered alpha-KTxs.

New "Birtoxin analogs" from Androctonus australis venom.

From the venom of the scorpion Androctonus australis, we have isolated a new bioactive polypeptide termed AaBTX-L1. When tested on the insect voltage-gated Na(+) channel (para) of the fruit fly, this toxin was able to induce a clear shift in activation (V(1/2)), resulting in the opening of the channel at more negative membrane potentials. Furthermore, AaBTX-L1 was totally devoid of toxicity when injected into mice intracerebroventricularly and did not compete with radiolabeled voltage-gated K(+) and Na(+) channel toxins in binding experiments on rat brain synaptosomes. Using its N-terminal amino acid sequence to design degenerate primers, several clones were amplified by PCR from the A. australis venom gland cDNA library. As a consequence, seven full oligonucleotide sequences encoding "long-chain" polypeptides with only three disulfide bridges have been cloned for the first time and are described here. Remarkably, they share high similarity with the anti-insect toxin Birtoxin from Parabuthus transvaalicus.