α-Conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors (original) (raw)
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Biochemical pharmacology, 2018
The ribbon isomer of α-conotoxin AuIB has 10-fold greater potency than the wild-type globular isomer at inhibiting nicotinic acetylcholine receptors (nAChRs) in rat parasympathetic neurons, and unlike its globular isoform, ribbon AuIB only targets a specific stoichiometry of the α3β4 nAChR subtype. Previous electrophysiological recordings of AuIB indicated that ribbon AuIB binds to the α3(+)α3(-) interface within the nAChR extracellular domain, which is displayed by the (α3)(β4) stoichiometry but not by (α3)(β4). This specificity for a particular stoichiometry is remarkable and suggests that ribbon isoforms of α-conotoxins might have great potential in drug design. In this study, we investigated the binding mode and structure-activity relationships of ribbon AuIB using a combination of molecular modeling and electrophysiology recording to determine the features that underpin its selectivity. An alanine scan showed that positions 4 and 9 of ribbon AuIB are the main determinants of th...
Marine Drugs, 2018
α-Conotoxins from Conus snails are capable of distinguishing muscle and neuronal nicotinic acetylcholine receptors (nAChRs). α-Conotoxin RgIA and αO-conotoxin GeXIVA, blocking neuronal α9α10 nAChR, are potential analgesics. Typically, α-conotoxins bind to the orthosteric sites for agonists/competitive antagonists, but αO-conotoxin GeXIVA was proposed to attach allosterically, judging by electrophysiological experiments on α9α10 nAChR. We decided to verify this conclusion by radioligand analysis in competition with α-bungarotoxin (αBgt) on the ligand-binding domain of the nAChR α9 subunit (α9 LBD), where, from the X-ray analysis, αBgt binds at the orthosteric site. A competition with αBgt was registered for GeXIVA and RgIA, IC50 values being in the micromolar range. However, high nonspecific binding of conotoxins (detected with their radioiodinated derivatives) to His6-resin attaching α9 LBD did not allow us to accurately measure IC50s. However, IC50s were measured for binding to Apl...
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.
Ribbon α-Conotoxin KTM Exhibits Potent Inhibition of Nicotinic Acetylcholine Receptors
Marine Drugs, 2019
KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3β2-nicotinic acetylcholine receptors (rα3β2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3β2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by α-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of...
Journal of Biological Chemistry, 2010
Non-native disulfide isomers of ␣-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of ␣-conotoxin AuIB have been characterized on ␣34 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to ϳ50%. The maximal inhibition was stoichiometry-dependent because altering ␣3:4 RNA injection ratios either increased AuIB(ribbon) efficacy (10␣:1) or completely abolished blockade (1␣:10). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity ␣34 nAChRs with corresponding EC 50 values of 22.6 and 176.9 M, respectively. Increasing the agonist concentration antagonized the inhibition of LS ␣34 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS ␣34 nAChRs by AuIB-(globular) was unaffected. Inhibition of LS and HS ␣34 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both ␣34 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS ␣34 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS ␣34 nAChRs competitively, whereas AuIB-(globular) inhibits ␣34 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.
Journal of Biological Chemistry, 2006
The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the ␣-subunit of nicotinic acetylcholine receptors and in particular the homomeric ␣7 nicotinic receptor. We report the isolation and characterization of an ␣-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the ␣7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the ␣32 nAChR indicating that ␣-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of ␣32 nAChRs. ␣-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17amino-acid, two-disulfide bridge peptide. The ligand is the first ␣-conotoxin with higher affinity for the closely related receptor subtypes, ␣32 versus ␣62, and selectively blocks these two subtypes when compared with ␣22, ␣42, and ␣11␦⑀ nAChRs.
The Journal of Neuroscience, 1998
Neuronal nicotinic acetylcholine receptors (nAChRs) with putative ␣34-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of ␣34-selective ligands. In this report, we describe the purification and characterization of an ␣34 nAChR antagonist, ␣-conotoxin AuIB, from the venom of the "court cone," Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. ␣-Conotoxin AuIB blocks ␣34 nAChRs expressed in Xenopus oocytes with an IC 50 of 0.75 M, a k on of 1.4 ϫ 10 6 min-1 M Ϫ1 , a k off of 0.48 min-1 , and a K d of 0.5 M. Furthermore, ␣-conotoxin AuIB blocks the ␣34 receptor with Ͼ100-fold higher potency than other receptor subunit combinations, including ␣22, ␣24, ␣32, ␣42, ␣44, and ␣11␥␦. Thus, AuIB is a novel, selective probe for ␣34 nAChRs. AuIB (1-5 M) blocks 20-35% of the nicotinestimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the ␣32specific ␣-conotoxin MII (100 nM) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, ␣34-containing nAChRs mediate norepinephrine release, whereas ␣32containing receptors mediate dopamine release.
Journal of Biological Chemistry, 2008
Nicotinic acetylcholine receptors (nAChRs) containing ␣3 and 2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related ␣6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog of ␣-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric ␣6/␣323 nAChRs with an IC 50 of 1.2 nM; in contrast, its IC 50 on the closely related ␣32 as well as non-␣6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the ␣3 subunit inserted into the homologous positions of the ␣6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the ␣6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the ␣3 subunit increased the IC 50 of the peptide by >1000-fold. Conversely, when these key residues in ␣3 were replaced with those from ␣6, the IC 50 decreased by almost 150-fold. Similar effects were seen with other ␣6-selective conotoxins, suggesting the general importance of these ␣6 residues in conferring selective binding.
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.
Journal of Biological Chemistry, 2006
The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the ␣-subunit of nicotinic acetylcholine receptors and in particular the homomeric ␣7 nicotinic receptor. We report the isolation and characterization of an ␣-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the ␣7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the ␣32 nAChR indicating that ␣-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of ␣32 nAChRs. ␣-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17amino-acid, two-disulfide bridge peptide. The ligand is the first ␣-conotoxin with higher affinity for the closely related receptor subtypes, ␣32 versus ␣62, and selectively blocks these two subtypes when compared with ␣22, ␣42, and ␣11␦⑀ nAChRs.