Elsevier

Biochemical Pharmacology

Volume 83, Issue 3, 1 February 2012, Pages 419-426
Biochemical Pharmacology

RegIIA: An α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs

https://doi.org/10.1016/j.bcp.2011.11.006Get rights and content

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes.

We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3β4 nAChRs found to date and is also active at α3β2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3β4 nAChRs.

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,

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