Mutagenesis of α-Conotoxins for Enhancing Activity and Selectivity for Nicotinic Acetylcholine Receptors (original) (raw)
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Conotoxin αD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors
Scientific reports, 2015
Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA, and is probably part ...
A New α-Conotoxin Which Targets α3β2 Nicotinic Acetylcholine Receptors
Journal of Biological Chemistry, 1996
We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of ␣32 subunits. This peptide, named ␣-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated ␣-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing ␣32 nAChRs with an IC 50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of ␣-conotoxin ImI, which selectively targets homomeric ␣7 neuronal nAChRs. Yet other ␣-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that ␣-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.
Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors
Acta Pharmacologica Sinica, 2009
Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson's disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.
α4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors
Peptides, 2008
Cone snails comprise approximately 500 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. α-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An α4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, α-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat α3β2 and α6α3β2 nAChRs. Comparing the distinct primary structure with other functionally related αconotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.
Marine drugs, 2017
Conotoxins are a pool of disulfide-rich peptide neurotoxins produced by cone snails for predation and defense. They are a rich reservoir of novel ligands for ion channels, neurotransmitter receptors and transporters in the nervous system. In this study, we identified a novel conotoxin component, O-conotoxin GeXXVIIA, from the venom of Conus generalis. The native form of this component is a disulfide-linked homodimer of a 5-Cys-containing peptide. Surprisingly, our electrophysiological studies showed that, in comparison to the folded monomers, the linear peptide of this toxin had the highest inhibitory activity at the human α9α10 nicotinic acetylcholine receptor (nAChR), with an IC50 of 16.2 ± 1.4 nM. The activities of the N-terminal and C-terminal halves of the linear toxin are markedly reduced compared with the full-length toxin, suggesting that the intact sequence is required to potently inhibit the hα9α10 nAChR. α9α10 nAChRs are expressed not only in the nervous system, but also ...
British journal of pharmacology, 2017
Neuronal α3-containing nicotinic acetylcholine receptors (nAChRs) in the peripheral nervous system (PNS) and non-neuronal tissues are implicated in a number of severe disease conditions ranging from cancer to cardiovascular diseases, and chronic pain. However, despite the physiological characterization of mouse models and cell lines, the precise pathophysiology of nAChRs outside the central nervous system (CNS) remains not well understood, in part because there is a lack of subtype-selective antagonists. α-Conotoxins isolated from cone snail venom exhibit characteristic individual selectivity profiles for nAChRs and, therefore, are excellent tools to study the determinants for nAChR-antagonist interactions. Given that human α3β4 subtype selective α-conotoxins are scarce and this is a major nAChR subtype in the PNS, the design of new peptides targeting this nAChR subtype is desirable. Recent studies using α-conotoxins RegIIA and AuIB, in combination with nAChR site-directed mutagenes...
Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview
Marine Drugs, 2014
Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium-(Na V ), potassium-(K V ), and calcium-(Ca V ) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure-function relationship data.
Differential Targeting of Nicotinic Acetylcholine Receptors by Novel αA-Conotoxins
Journal of Biological Chemistry, 1997
We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12-18 residue, 4 Cys-containing ␣-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named ␣A-conotoxins EIVA and EIVB, block both Torpedo and mouse ␣1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to ␣-bungarotoxin, ␣A-EIVA is inactive at ␣7-containing nAChRs even at micromolar concentrations. In this regard, ␣A-EIVA is similar to the previously described ␣-conotoxins (e.g. ␣-MI and ␣-GI) which also selectively target ␣1versus ␣7-containing nAChRs. However, ␣-MI and ␣-GI discriminate between the ␣/␦ versus ␣/␥ subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, ␣A-conotoxin EIVA blocks both the ␣/␥ site and ␣/␦ site with equally high affinity but with distinct kinetics. The ␣A-conotoxins thus represent novel probes for the ␣/␥ as well as the ␣/␦ binding sites of the nAChR.
alpha.-Conotoxin EI, A New Nicotinic Acetylcholine Receptor Antagonist with Novel Selectivity
Biochemistry, 1995
We report the isolation and characterization of a novel nicotinic acetylcholine receptor (nAChR) ligand. The toxin is an 18 amino acid peptide and is the first reported a-conotoxin from an Atlantic fish-hunting Conus. The peptide was purified from the venom of Conus emineus and is called a-conotoxin EI. The sequence diverges from that of previously isolated a-conotoxins. We demonstrate that this structural divergence has functional consequences. In Torpedo nAChRs, a-conotoxin EI selectively binds the agonist site near the a/d subunit interface in contrast to a-conotoxin MI which selectively targets the a / y agonist binding site. In mammalian nAChRs a-conotoxin EI shows high affinity for both the a/d and a / y subunit interfaces (with some preference for the a/d site), whereas a-conotoxin MI is highly selective for the d d ligand binding site. The sequence of the peptide is: Arg-Asp-Hyp-Cys-Cys-Tyr-His-Pro-Thr-Cys-Asn-Met-Ser-Asn-Pro-Gln-Ile-Cys-NH~, with disulfide bridging between Cys4-CyslO and CysS-Cysl8, analogous to those of previously described a-conotoxins. This sequence has been verified by total chemical synthesis. Thus, a-conotoxin EI is a newly-available tool with unique structure and function for characterization of nAChRs. Nicotinic acetylcholine receptors (nAChRs)' in skeletal muscle and the electric organ of Torpedo are heteropentameric ligand-gated cation channels formed by four subunits in the stoichiometry (al)& 76. Several small molecule toxins isolated from plants, coral, and gastropods as well as polypeptide toxins from predatory organisms have been isolated which target nAChRs [for review see Chiappinelli (1993)l. The availability of these toxins has played a critical role in the progressive understanding of the structure and function of the nicotinic receptor. The nAChR requires two molecules of acetylcholine to bind two separate sites for channel opening. These nonequivalent binding sites are located at the a l y and al6 subunit interfaces (Blount & Merlie, 1989). Curariform antagonists bind with 1-2 orders of magnitude higher affinity to the a l y site than to the al6 site of both mammalian muscle and Torpedo receptors + This work was supported by NIMH Scientist Development Award for Clinicians K20 MHO0929 (J.M.M.), NIH Grants GM 48677 (B.M.O.) and NS 29951 (S.N.A.), and the Smokeless Tobacco Research Council (S.N.A.).
Biochemistry
We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, RS-conotoxin RVIIIA (RS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, RS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, σ-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT 3 receptor; thus, RS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the RS-conotoxin family to target the muscle nAChR and paralyze prey.