RegIIA: An α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs
Graphical abstract
Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) play an important role in the central and peripheral nervous system and are implicated in certain disease states including Parkinson's disease, schizophrenia, depression, Alzheimer's disease, and nicotine addiction [1]. These receptors are nicotine-sensitive ligand gated ion channels that are endogenously activated by acetylcholine. Structurally, most neuronal nAChRs are heteropentamers of α2-6 and β2-4 subunits combined in different stoichiometries [2]. There are homomeric nAChRs, notable among these is the α7 nAChR, and heteromeric receptors that are composed of only alpha subunits such as α9α10 nAChRs.
The specific combinations of α and β subunits mediate the diverse population of neuronal nAChRs subtypes with different subtypes having a specific function and distribution in the central and peripheral nervous system. α4β2 nAChRs are the predominant subtype in the brain, they have the highest affinity for nicotine (Ki = 0.6–10 nM) and account for >90% of binding of nicotine in brain tissues [3]. Transgenic knockout of α4 or β2 subunits eliminate nicotine self-administration in mice. Re-instatement of these subunits in the knockout restores nicotine self-administration, implicating this receptor in nicotine addiction [4].
Other nAChR subtypes, particularly the α3β4, can also be involved in addiction of nicotine and other drugs of abuse [4]. The α3β4 is the predominant nAChR in the sensory and autonomic ganglia and in subpopulations of CNS neurons, such as medial habenula and dorsal medulla [2]. This receptor is involved in the mesolimbic dopamine pathway and is thought to be important in certain feedback rewarding effects under a substance abuse regimen. Furthermore, nicotine-induced hypolocomotion is reduced in β4 null mice, thus emphasizing the importance of α3β4 in the nicotine related effects in the CNS [5]. While the involvement of α3β4 receptors in psychostimulant and drug-abusive behavior has been established [6], the lack of adequate molecular probes that allow exploring the neurophysiology of these receptors is a limiting factor for establishing their precise role in addiction.
α-Conotoxins, ubiquitous compounds found in the venoms of cone snails [7], are short disulfide-constrained peptides that target various nAChR subtypes. α-Conotoxin sequences have four cysteines arranged in CC–Xn–C–Ym–C pattern, where Xn is a loop of amino acids with n = 3–4 and Ym is a loop of amino acids with m = 3–7. The sizes of the loops are used for α-conotoxin classification (i.e., an α4/7-conotoxin has 4 residues in the X loop and 7 residues in the Y loop). The number and nature of the amino acids in these loops are defining for the binding and selectivity of α-conotoxins towards nAChRs subtypes. The sequences of α-conotoxins are species-specific, therefore, the discovery of new α-conotoxins can provide new tools for the functional exploration of nAChR subtypes. In general, α-conotoxins target more than one nAChR subtype; however, their selectivity and potency can vary widely. Here we describe the discovery, together with the biochemical, biophysical and functional characterization of RegIIA, an α4/7-conotoxin isolated from the venom of Conus regius, a worm-hunting cone snail species that inhabits the Western Atlantic Ocean. RegIIA is among the most potent antagonist of α3β4 nAChRs to date, and it does not inhibit the α4β2 subtype. This selectivity profile makes RegIIA a prospective probe for studying nicotine addiction processes. RegIIA has a classical α-conotoxin globular structure (ω-shaped fold) indicating that it has an exquisite balance of shape, charges, and polarity exposed on its surface to enable it to potently block the α3β4 nAChR.
Section snippets
Specimen collection, RegIIA isolation and characterization
Specimens of C. regius (35–70 mm in length) were collected off the Florida Keys (Plantation Key), USA, using SCUBA at depths ranging from 2 to 10 m. Venom ducts dissected from specimens of C. regius were homogenized in 0.1% TFA (Fisher Scientific, PA) at 4 °C. Whole extracts were centrifuged at 10,000 × g for 20 min, at 4 °C, and the resulting pellets were washed three times with 0.1% TFA and re-centrifuged under identical conditions. The supernatants containing the soluble peptides were pooled,
Discovery, characterization and synthesis of RegIIA
Fractionation of the venom of C. regius (SE and RP-HPLC) produced a pure peptide with a monoisotopic molecular mass of 1664.9 Da (Fig. 1). The amino acid sequence of the peptide (RegIIA) was obtained by Edman degradation (Fig. 1, Table 1), and corresponds to a 16-residue/2-disulfide bond hydrophilic conopeptide without aromatic residues, which has a sequence homology to other α4/7-conotoxins (Table 1). RegIIA was synthesized with all l-amino acids. NMR comparison of the native material with the
Discussion
Here we describe the isolation and characterization of RegIIA, an α4/7-conotoxin from the venom a C. regius, a Western Atlantic worm-hunting cone snail species. This conotoxin, initially described as reg2a [27], has a sequence remarkably similar to OmIA [28], an α-conotoxin from Conus omaria, an Indo-Pacific mollusk-hunting species. These two conotoxins only differ in presence of an additional Gly residue at the C-terminal in OmIA. RegIIA has also significant sequence homology with GIC [29] and
Acknowledgments
This research was supported by the National Institutes of Health (NIH) Grants 1R15GM066004-01A1 and 1R21NS066371-01 (FM); The Florida Seagrant program Grant R/LR-MB-28 (FM); Australian Research Council Discovery Project Grant (DJA, DJC and FM); Australian Research Council Professorial Fellowship (DJA); Australian National Health and Medical Research Grant (PFA and KBA); “LOEWE–Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse‘s Ministry of Higher Education,
References (60)
- et al.
Diversity of vertebrate nicotinic acetylcholine receptors
Neuropharmacology
(2009) Multiple brain pathways and receptors underlying tobacco addiction
Biochem Pharmacol
(2007)- et al.
The α3 and β4 nicotinic acetylcholine receptor subunits are necessary for nicotine-induced seizures and hypolocomotion in mice
Neuropharmacology
(2004) - et al.
The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: dual role in nicotine addiction and lung cancer
Prog Neurobiol
(2010) - et al.
Direct cDNA cloning of novel conopeptide precursors of the O-superfamily
Peptides (New York, NY, United States)
(2005) - et al.
cDNA cloning of two A-superfamily conotoxins from Conus striatus
Toxicon
(2003) - et al.
α-Conotoxins PnIA and [A10L]PnIA stabilize different states of the α7-L247T nicotinic acetylcholine receptor
J Biol Chem
(2003) - et al.
Inhibition of neuronal nicotinic acetylcholine receptor subtypes by α-conotoxin GID and analogues
J Biol Chem
(2009) - et al.
The A-superfamily of conotoxins: structural and functional divergence
J Biol Chem
(2004) - et al.
The synthesis, structural characterization, and receptor specificity of the α-conotoxin Vc1.1
J Biol Chem
(2006)
Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist α-conotoxin OmIA that discriminates α3 vs α6 nAChR subtypes
Biochem Biophys Res Commun
α-Conotoxin OmIA Is a potent ligand for the acetylcholine-binding protein as well as α3β2 and α7 nicotinic acetylcholine receptors
J Biol Chem
α-Conotoxin GIC from Conus geographus, a novel peptide antagonist of nicotinic acetylcholine receptors
J Biol Chem
Isolation, structure, and activity of GID, a novel α4/7-conotoxin with an extended N-terminal sequence
J Biol Chem
α-Conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors
J Biol Chem
The prodomain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors
J Biol Chem
Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins
J Biol Chem
A novel α-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat α9α10 and α7 nicotinic cholinergic receptors
J Biol Chem
α-Conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors
J Biol Chem
A novel mechanism of inhibition of high-voltage activated calcium channels by α-conotoxins contributes to relief of nerve injury-induced neuropathic pain
Pain
Structure and activity of α-conotoxin PeIA at nicotinic acetylcholine receptor subtypes and GABAB receptor-coupled N-type calcium channels
J Biol Chem
Embryonic toxin expression in the cone snail Conus victoriae—primed to kill or divergent function?
J Biol Chem
Single amino acid substitutions in κ-conotoxin PVIIA disrupt interaction with the Shaker K+ channel
J Biol Chem
Single amino acid substitutions in α-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor
J Biol Chem
α-Conotoxins ImI and ImII similar α7 nicotinic receptor antagonists act at different sites
J Biol Chem
Atypical α-conotoxin LtIA from Conus litteratus targets a novel microsite of the α3β2 nicotinic receptor
J Biol Chem
α-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors
J Biol Chem
Antagonism of α3β4 nicotinic receptors as a strategy to reduce opioid and stimulant self-administration
Eur J Pharmacol
Mammalian nicotinic acetylcholine receptors: from structure to function
Physiol Rev
Nicotine activation of α4* receptors: sufficient for reward, tolerance, and sensitization
Science
Cited by (54)
Biomedical applications of synthetic peptides derived from venom of animal origin: A systematic review
2024, Biomedicine and PharmacotherapyMarine-derived nicotinic receptor antagonist toxins: Pinnatoxins and alpha conotoxins
2021, Advances in NeurotoxicologyCitation Excerpt :The reverse is also true. α-CTx OmIA from C. omaria, and α-CTx RegIIA, from C. regius, both block α3β2 with higher potency than α7, but are only 3–20-fold more potent at α3β2 (Chi et al., 2006; Franco et al., 2012; Talley et al., 2006). There are some conotoxins with higher selectivity toward α3β2 receptors relative to α7, however they are still not completely devoid of α7 nAChR activity.