α-Conotoxin VnIB from Conus ventricosus is a potent and selective antagonist of α6β4* nicotinic acetylcholine receptors

Highlights

• α-Conotoxin VnIB is a newly identified peptide from Conus ventricosus.

• Unlike other native α-conotoxins, VnIB selectively inhibits α6β4 nAChRs.

• VnIB selectivity includes the closely related α6β2β3 and α3β4 nAChR subtypes.

• VnIB exhibits rapid binding and unbinding at the rat α6β4 nAChR.

• VnIB is a novel probe for elucidation of the structure and function of α6β4* nAChRs.

Abstract

α6-containing (α6*) nicotinic acetylcholine receptors (nAChRs) are expressed throughout the periphery and the central nervous system and constitute putative therapeutic targets in pain, addiction and movement disorders. The α6β2* nAChRs are relatively well studied, in part due to the availability of target specific α-conotoxins (α-Ctxs). In contrast, all native α-Ctxs identified that potently block α6β4 nAChRs exhibit higher potencies for the closely related α6β2β3 and/or α3β4 subtypes. In this study, we have identified a novel peptide from Conus ventricosus with pronounced selectivity for the α6β4 nAChR. The peptide-encoding gene was cloned from genomic DNA and the predicted mature peptide, α-Ctx VnIB, was synthesized. The functional properties of VnIB were characterized at rat and human nAChRs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. VnIB potently inhibited ACh-evoked currents at rα6β4 and rα6/α3β4 nAChRs, displayed ∼20-fold and ∼250-fold lower potencies at rα3β4 and rα6/α3β2β3 receptors, respectively, and exhibited negligible effects at eight other nAChR subtypes. Interestingly, even higher degrees of selectivity were observed for hα6/α3β4 over hα6/α3β2β3 and hα3β4 receptors. Finally, VnIB displayed fast binding kinetics at rα6/α3β4 (on-rate t_½ = 0.87 min⁻¹, off-rate t_½ = 2.7 min⁻¹). The overall preference of VnIB for β4* over β2* nAChRs is similar to the selectivity profiles of other 4/6 α-Ctxs. However, in contrast to previously identified native α-Ctxs targeting α6* nAChRs, VnIB displays pronounced selectivity for α6β4 nAChRs over both α3β4 and α6β2β3 receptors. VnIB thus represents a novel molecular probe for elucidating the physiological role and therapeutic properties of α6β4* nAChRs.

Introduction

The neurotransmitter acetylcholine (ACh) mediates its fast neurotransmission through a highly heterogeneous family of ligand-gated ion channels, the nicotinic ACh receptors (nAChRs) (Albuquerque et al., 2009, Taly et al., 2009). The nAChRs are expressed throughout the periphery and the central nervous system (CNS), where they are involved in numerous distinct physiological functions, and the receptors have been targets for drug development for a wide range of neurological, psychiatric and cognitive disorders as well as for various forms of pain and addiction (Bertrand et al., 2015, Bertrand and Terry, 2018, Hone et al., 2018, Picciotto et al., 2015, Quik and Wonnacott, 2011). A prominent recent example is the nAChR agonist varenicline, a smoking cessation drug with annual global sales in 2018 of $1085 million (Pfizer Inc., 2019).

The nAChRs belong to the Cys-loop receptor superfamily, which also includes the 5-HT₃ serotonin, γ-aminobutyric acid_A and the glycine receptors (Chua and Chebib, 2017, Lynch et al., 2017, Walstab et al., 2010). Like other members of this receptor family, nAChRs are composed of five subunits surrounding a central ion pore. Signal transduction through the nAChR is initiated by agonist binding to orthosteric sites located in the extracellular subunit interfaces in the pentameric complex, which triggers the opening of the central ion pore, thus enabling cation influx into and depolarization of the neuron (Albuquerque et al., 2009, Taly et al., 2009).

The heterogeneity of native nAChRs arises from the existence of a total of 17 receptor subunits. In mammals, neuronal nAChRs are composed of different combinations of α (α2-7, α9 and α10) and β (β2-4) subunits, which assemble to form homomeric (composed exclusively of α7 or α9 subunits) or heteromeric (various α2-6/β2-4 combinations, α7β2 and α9α10) receptor complexes (Albuquerque et al., 2009, Taly et al., 2009). This gives rise to a wide range of receptor subtypes with distinct functional properties and expression patterns in vivo. Therefore, subtype-selective ligands, capable of discriminating between this multitude of receptor subtypes, are crucial for studies of the specific physiological roles of individual nAChRs.

The α4β2* and α7* (asterisk indicates the possible presence of additional subunits) nAChRs are the most abundant nAChR subtypes and are essentially expressed in all CNS regions (Taly et al., 2009). In contrast, α6* nAChRs display a much more restricted CNS distribution, as these receptors are confined mainly to the catecholaminergic pathways and the visual system (Azam et al., 2002, Le Novère et al., 1996). The α6 subunit forms functional receptors in combination with α4, β2, β3 and/or β4 subunits in vivo (Kuryatov et al., 2000, Letchworth and Whiteaker, 2011). α6β2* receptors (α6β2, α6β2β3, α4α6β2β3) are the predominant α6* nAChRs in the visual system (Gotti et al., 2005, Quik et al., 2011) and have been demonstrated to be involved in modulation of dopamine release from the striatum and nucleus accumbens (Azam et al., 2002, Letchworth and Whiteaker, 2011). These findings have sparked considerable interest in these receptors as potential therapeutic targets in Parkinson's disease and nicotine addiction (Champtiaux et al., 2002, Miwa et al., 2011, Quik and Wonnacott, 2011). In contrast, albeit less studied, α6β4* nAChRs have been reported to regulate exocytosis in human and monkey adrenal chromaffin cells (Hernández-Vivanco et al., 2014, Pérez-Alvarez et al., 2012), to control the release of norepinephrine in mouse hippocampus (Azam and McIntosh, 2006), and to be expressed in rodent dorsal root ganglia neurons, where they may be involved in pain signaling (Hone et al., 2012a, Wieskopf et al., 2015).

Predatory marine snails of the Conus species feeds on mollusks, marine worms and fish and utilize their complex venoms to immobilize and capture their prey (Olivera et al., 2008). The genes encoding the toxins can be grouped into superfamilies based on similarities in the highly conserved signal sequence region of the peptide precursor. The mature conopeptides encoded by these genes can be characterized by the arrangements of their cysteine residues and by their pharmacological activity (Kaas et al., 2012, Robinson and Norton, 2014). α-Conotoxins (α-Ctx) are the predominant members of the A-superfamily of genes; these disulfide-rich peptides specifically target different nAChR subtypes and generally have a framework I cysteine pattern of CC-C-C with a Cys₁-Cys₃ and Cys₂-Cys₄ disulfide bond connectivity (Abraham and Lewis, 2018). α-Ctxs can be further subdivided based on the number of non-cysteine residues in the first and second inter-cysteine loop. The 3/5 α-Ctxs (three residues in the first cysteine-loop, five in the second) are selective blockers of the muscle nAChRs, while the 4/3, 4/4, 4/6 and 4/7 α-Ctxs mainly target neuronal nAChRs (Azam and McIntosh, 2009, Terlau and Olivera, 2004). Other members of the A-superfamily possess the same signal sequence homology, but display a framework IV cysteine pattern (CC–C–C–C–C) and target either voltage-gated K⁺ or Na⁺ channels (Craig et al., 1998, Kelley et al., 2006, Santos et al., 2004, Teichert et al., 2007).

Due to their pronounced selectivity for specific nAChR subtypes, α-Ctxs have been used in a large variety of studies to elucidate the physiological functions governed by different neuronal and muscle-type nAChRs (reviewed in (Giribaldi and Dutertre, 2018, Lebbe et al., 2014)). Relatively few of the α-Ctxs published to date target α6* nAChRs (Azam et al., 2005, Cartier et al., 1996, Dowell et al., 2003, Luo et al., 2013a, Luo et al., 2013b). α-Ctx MII was the first α-Ctx found to potently block the α6β2β3 receptor (tested using the α6/α3 chimera as a surrogate α6 subunit) and the closely related α3β2 nAChR with comparable nanomolar antagonist potencies (Cartier et al., 1996). In contrast, both α-Ctxs PIA and BuIA inhibit α6β2β3-signaling more potently than the signaling of α6β4 and α3β2, and this has made them valuable tools in studies of α6β2* nAChRs (Azam et al., 2005, Dowell et al., 2003). Interestingly, there are no reports to date of native α-Ctxs that preferentially target α6β4* nAChRs over the closely related α6β2* and α3β4* subtypes (Azam et al., 2005, Dowell et al., 2003, Luo et al., 2013b, Smith et al., 2013). In the present study we report the cloning, synthesis and pharmacological characterization of a novel α-Ctx, VnIB, from the venom of Conus ventricosus, which is found in shallow waters of the Mediterranean and hunts polychaete worms (Romeo et al., 2008). α-Ctx VnIB is the first native α-Ctx that potently and preferentially antagonizes the signaling through the neuronal α6β4 nAChR.