Biomedical subjects

Stéphanie Mouhat

Publications and source records attributed to Stéphanie Mouhat.

5 recordsLinked to original sources

PubMed2005

Pharmacological profiling of Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal domain.

OSK1, a toxin from the venom of the Asian scorpion Orthochirus scrobiculosus, is a 38-residue peptide cross-linked by three disulfide bridges. A structural analog of OSK1, [Lys(16),Asp(20)]-OSK1, was found previously to be one of the most potent blockers of the voltage-gated K(+) channel Kv1.3 hitherto characterized. Here, we demonstrate that progressive trimming of the N-terminal domain of [Lys(16),Asp(20)]-OSK1 results in marked changes in its pharmacological profile, in terms of both K(+) channel affinity and selectivity. Whereas the affinity to Kv1.1 and Kv1.3 did not change significantly, the affinity to Kv1.2 and K(Ca)3.1 was drastically reduced with the truncations. It is surprising that a striking gain in potency was observed for Kv3.2. In contrast, a truncation of the C-terminal domain, expected to partially disrupt the toxin beta-sheet structure, resulted in a significant decrease or a complete loss of activity on all channel types tested. These data highlight the value of structure-function studies on the extended N-terminal domain of [Lys(16),Asp(20)]-OSK1 to identify new analogs with unique pharmacological properties.

Amino Acid Sequence↗

PubMed2005

K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom.

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges that was initially isolated from the venom of the Asian scorpion Orthochirus scrobiculosus. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+-activated K+ channels in vitro. In the present paper, we report that OSK1 is lethal in mice by intracerebroventricular injection, with a LD50 (50% lethal dose) value of 2 microg/kg. OSK1 blocks K(v)1.1, K(v)1.2, K(v)1.3 channels potently and K(Ca)3.1 channel moderately, with IC50 values of 0.6, 5.4, 0.014 and 225 nM respectively. Structural analogues of OSK1, in which we mutated positions 16 (Glu16-->Lys) and/or 20 (Lys20-->Asp) to amino acid residues that are conserved in all other members of the alpha-KTx3 toxin family except OSK1, were also produced and tested. Among the OSK1 analogues, [K16,D20]-OSK1 (OSK1 with Glu16-->Lys and Lys20-->Asp mutations) shows an increased potency on K(v)1.3 channel, with an IC50 value of 0.003 nM, without loss of activity on K(Ca)3.1 channel. These data suggest that OSK1 or [K16,D20]-OSK1 could serve as leads for the design and production of new immunosuppressive drugs.

Amino Acid Sequence↗

PubMed2004

Toxin determinants required for interaction with voltage-gated K+ channels.

Ion channel-acting toxins are mainly short peptides generally present in minute amounts in the venoms of diverse animal species such as scorpions, snakes, spiders, marine cone snails and sea anemones. Interestingly, these peptides have evolved over time on the basis of clearly distinct architectural motifs present throughout the animal kingdom, but display convergent molecular determinants and functional homologies. As a consequence of this conservation of some key determinants, it has also been evidenced that toxin targets display some common evolutionary origins. Indeed, these peptides often target ion channels and ligand-gated receptors, though other interacting molecules such as enzymes have been further evidenced. In this review, we provide an overview of some selected peptides from various animal species that act on specific K+ conducting voltage-gated ion channels. In particular, we emphasize our global analysis on the structural determinants of these molecules that are required for the recognition of a particular ion channel pore structure, a property that should be correlated to the blocking efficacy of the K+ efflux out of the cell during channel opening. A better understanding of these molecular determinants is valuable to better specify and derive useful peptide pharmacological properties.

Models, Chemical↗

PubMed2004

Diversity of folds in animal toxins acting on ion channels.

Animal toxins acting on ion channels of excitable cells are principally highly potent short peptides that are present in limited amounts in the venoms of various unrelated species, such as scorpions, snakes, sea anemones, spiders, insects, marine cone snails and worms. These toxins have been used extensively as invaluable biochemical and pharmacological tools to characterize and discriminate between the various ion channel types that differ in ionic selectivity, structure and/or cell function. Alongside the huge molecular and functional diversity of ion channels, a no less impressive structural diversity of animal toxins has been indicated by the discovery of an increasing number of polypeptide folds that are able to target these ion channels. Indeed, it appears that these peptide toxins have evolved over time on the basis of clearly distinct architectural motifs, in order to adapt to different ion channel modulating strategies (pore blockers compared with gating modifiers). Herein, we provide an up-to-date overview of the various types of fold from animal toxins that act on ion channels selective for K+, Na+, Ca2+ or Cl- ions, with special emphasis on disulphide bridge frameworks and structural motifs associated with these peptide folds.

Amino Acid Sequence↗

PubMed2004

The 'functional' dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels.

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 microg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 microM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

Amino Acid Sequence↗