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Venom‐derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage‐gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV1.7 and hCaV3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay‐guided HPLC fractionation revealed a 36‐amino acid peptide with three disulfide bridges named μ/ω‐theraphotoxin‐Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV1.7 and hCaV3. Nuclear magnetic resonance structure determination of Pmu1a shows an inhibitor cystine knot fold that is the characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV3.2 and hNaV1.7 voltage‐gated channels. [ABSTRACT FROM AUTHOR]

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85 μ m) while inhibiting apoptosis (EC50 2.2 μ m). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors. [ABSTRACT FROM AUTHOR]

The glycopeptide CcTx, isolated from the venom of the piscivorous cone snail Conus consors, belongs to the κA-family of conopeptides. These toxins elicit excitotoxic responses in the prey by acting on voltage-gated sodium channels. The structure of CcTx, a first in the κA-family, has been determined by high-resolution NMR spectroscopy together with the analysis of its O-glycan at Ser7. A new type of glycopeptide O-glycan core structure, here registered as core type 9, containing two terminal L-galactose units {α- L-Gal p-(1→4)-α- D-Glc pNAc-(1→6)-[α- L-Gal p-(1→2)-β- D-Gal p-(1→3)-]α- D-Gal pNAc-(1→O)}, is highlighted. A sequence comparison to other putative members of the κA-family suggests that O-linked glycosylation might be more common than previously thought. This observation alone underlines the requirement for more careful and in-depth investigations into this type of post-translational modification in conotoxins. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 38-47. Abstract Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α1 subunit by the corresponding residues of the β subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α1 GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ββ-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites. [ABSTRACT FROM AUTHOR]

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases. [ABSTRACT FROM AUTHOR]

Abstract: Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of l-Phe to d-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture. [Copyright &y& Elsevier]

α-Conotoxins, from cone snails, and α-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered α-neurotoxins to be docked onto the α7 nAChR. To investigate if α-conotoxins interact with the nAChR in a similar manner, we built homology models of human α7 and α3 β2 nAChRs, and performed docking simulations of α-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that α-conotoxins have a different mode of interaction compared with α-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the β9/ β10 hairpin of the (+)α7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on α7 or α3 β2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish α-conotoxin and α-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Recent and increasing use of computational tools in the field of nicotinic receptors has led to the publication of several models of ligand–receptor interactions. These models are all based on the crystal structure at 2.7 Å resolution of a protein related to the extracellular N-terminus of nicotinic acetylcholine receptors (nAChRs), the acetylcholine binding protein. In the absence of any X-ray or NMR information on nAChRs, this new structure has provided a reliable alternative to study the nAChR structure. We are now able to build homology models of the binding domain of any nAChR subtype and fit in different ligands using docking programs. This strategy has already been performed successfully for the docking of several nAChR agonists and antagonists. This minireview focuses on the interaction of α-conotoxins with neuronal nicotinic receptors in light of our new understanding of the receptor structure. Computational tools are expected to reveal the molecular recognition mechanisms that govern the interaction between α-conotoxins and neuronal nAChRs at the molecular level. An accurate determination of their binding modes on the neuronal nAChR may allow the rational design of α-conotoxin-based ligands with novel nAChR selectivity. [ABSTRACT FROM AUTHOR]