Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview

Abstract

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.

1. Cone Snails, the New Gold Mines?

In general, venom peptides offer a unique and extensive source of chemical diversity as they are driven by evolutionary pressure to improve prey capture and/or protection of the species. This chemical diversity can be found in animals as diverse as sea anemones, jellyfish, spiders, scorpions, cone snails, etc. [1]. Among these species, venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds that are increasingly recognized as an emerging source of peptide-based therapeutics. Their ability to use a diverse array of small disulfide-bridged peptides (conopeptides or conotoxins) for prey capture makes them unique. Moreover, they are considered as specialized predators which have developed the most sophisticated peptide chemistry and neuropharmacology for their own biological purposes by producing venoms that contain a structural and functional variety of neurotoxins.

Conotoxins display a great molecular diversity, being evolved across all phylogenetic clades and feeding strategies of cone snails. This multiplicity is mirrored in the classification of at least 16 genetically distinct superfamilies where the conotoxins are categorized upon their cysteine-framework. These superfamilies are subdivided in conotoxin families depending on their impressive diversity of targets ranging from voltage-gated ion channels (sodium, potassium, and calcium) to ligand-gated ion channels (such as nicotine receptors and serotonin receptors). The implementation of this broad spectrum of pharmacologically active components has made this single genus very successful, evolving into more than 500 Conus species [2]. Each cone snail species produces more than 1000 conopeptides with an estimated overlap of 5% between different species [3]. To date, only 0.1% out of potentially 500,000 venom components has been functionally and structurally investigated. Nevertheless, the consideration of Conus venoms as gold mines for the discovery of new therapeutics is validated by the knowledge that, out of the limited number of studied conopeptides, already six peptides have reached human clinical trials, and one was approved as analgesic in 2004. The toxins of Conus sp. are usually potent, selective and small (typically <5 kDa) which is an advantage for cost-effective synthesis and makes them ideal pharmacological probes [4].

This review will focus on one conotoxin family in particular, namely the α-conotoxins. These toxins are nicotinic acetylcholine receptor (nAChR) antagonists that are used by the cone snails to immobilize their prey. Here, we discuss the structure–function relationship and molecular pharmacology of α-conotoxins specifically interacting with nAChRs.

2. Alpha-Conotoxins, the Largest Characterized Group of Conotoxins

Conus species have evolved multiple classes of conopeptides targeting ligand-gated ion channels including nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine3 receptors (5-HT₃Rs), and N-methyl-d-aspartate (NMDA) antagonists as well as α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) enhancers. Among these receptor classes, antagonists of nAChRs are the largest and most diverse. Moreover, along with the NMDA antagonists, they show the highest potential as lead compounds to new ligand-gated ion channel therapeutics [5].

In almost every Conus venom investigated until now, at least one conotoxin that inhibits nAChRs was found [6,7]. Because many of the known prey of Conus use cholinergic transmission at their neuromuscular junctions, it is believed that the venom of each cone snail species contains at least one nAChR antagonist. The great majority of the >500 species of cone snails paralyzes polychaete worms, others paralyze mollusks and various invertebrates such as echiuroid worms and hemichordates. A minority use their venom to prey on fish. Each cone snail species is specialized because they often eat exclusively one prey species [8]. Overall, seven different families of conotoxins are known to target nAChRs. The largest group of characterized Conus sp. peptides is the family of α-conotoxins (belonging to the A-superfamily), that are selective antagonists of the muscle and neuronal subtype nAChRs [6]. They act at the nAChR acetylcholine binding site as competitive antagonists and are among the smallest of the conopeptides (12–20 amino acid residues) [6,9]. Alpha-conotoxins have a characteristic CC-X_m-C-X_n-C framework, where the four cysteines can yield three possible disulfide connectivities: globular (I–III, II–IV), ribbon (I–IV, II–III) and beads (I–II, III–IV). However, naturally appearing α-conotoxins typically exhibit the globular conformation [10]. The number of residues included within the two loops (m,n) of α-conotoxins is the basis for the division into several structural subgroups (m/n: 3/5, 4/3, 4/4, 4/5, 4/6 and 4/7). The loop size is believed to roughly correlate with the pharmacological target selectivity. In general, α-conotoxins with a 3/5 framework are isolated from fish-hunting snails and are active toward fish and/or mammalian neuromuscular nAChRs, whereas conotoxins from the 4/3, 4/4, 4/5, 4/6 or 4/7 classes mainly interact with mammalian neuronal nAChRs [9]. The most commonly reported framework is the 4/7 subgroup. Within this subgroup, an interesting α-conotoxin is Vc1.1 which potently inhibits neuronal (α₃, α₅, α₇, β₄ and α₉α₁₀ nAChR subunits) versus muscle nAChRs [11,12] (Figure 1). Therefore, it was selected for tests in pain models revealing Vc1.1 as the first α-conotoxin with an analgesic effect [13,14].

Figure 1. α-conotoxin Vc1.1 three-dimensional NMR solution structure (PDB:2H8S) and amino acid sequence representation with indication of the two disulfide bonds. Figure was prepared using the program PyMOL [15].

Figure 1. α-conotoxin Vc1.1 three-dimensional NMR solution structure (PDB:2H8S) and amino acid sequence representation with indication of the two disulfide bonds. Figure was prepared using the program PyMOL [15].

The gene structure of A-superfamily conotoxins is unique as it is the only superfamily having one intron in between two exons, while most conotoxin superfamily genes contain two introns [16]. In general, members from the same superfamily share a highly conserved signal peptide (pre-region) whereas the pro-region is less preserved. In α-conotoxins, the large intron can be found in this pro-region. Finally, the C-terminal toxin-encoding region is highly variable [16,17].

Alpha-conotoxins that are selective for a specific nAChR significantly contributed to their characterization both in vivo and in vitro, and some of these specific peptides may possess therapeutic potential [11]. The pharmacophore of these α-conotoxins has been investigated in detail. It is composed of a conserved hydrophobic patch in the first loop which determines binding, and a more variable second loop, which administers selectivity through pairwise interactions with different nAChRs subunits [18]. In this way, the selectivity of α-conotoxins isolated from different Conus species not only contributed to their characterization but also enabled the dissection of the functional roles of nAChR subtypes [6,9].

3. Nicotinic Acetylcholine Receptors (nAChRs)

Chemical signaling in the central and peripheral nervous systems is mediated by rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). This ion channel family includes nicotinic acetylcholine (nAChRs), serotonin-type-3 (5-HT₃Rs), γ-aminobutyric acid-A (GABA_ARs), and glycine receptors (GlyRs) [19]. All these receptors exist in at least three distinct states which are interconvertible: resting (unliganded, closed channel), activated (liganded, open channel), and desensitized (liganded, closed channels). The binding of agonists, antagonists and allosteric drugs alters the equilibria between these interconvertible states. Cys-loop LGICs are compiled of five identical or homologous subunits arranged pseudosymmetrically around a central ion-conducting channel, like staves around a barrel. When a neurotransmitter binds in the extracellular ligand-binding domain, rapid opening of an intrinsic ion channel in the transmembrane domain of the receptor is triggered. With prolonged neurotransmitter exposure, the channel shifts to a non-conducting desensitized state [20].

Nicotinic acetylcholine receptors, being a member of ligand-gated cationic channels, mediate fast synaptic transmission. They are broadly distributed throughout the peripheral and central nervous systems of both simple and evolutionarily complex organisms [21]. As these structures are highly conserved over a wide range of species, the importance of nAChRs in the nervous system cannot be neglected. Moreover, this general appearance also provides a platform for translational research from in vitro ligand discovery to in vivo characterization in various animal models of human diseases [22]. Examples of these diseases include central nervous system (CNS) disorders such as epilepsy, Alzheimer’s disease, Parkinson’s disease, schizophrenia, nicotine addiction, pain, cancer, etc. [23,24,25,26]. The contribution of nAChRs disorders to the above mentioned pathophysiologic states can be found in the fact that presynaptic nAChRs induce various brain regions to release several neurotransmitters, including dopamine, norepinephrine, serotonin and acetylcholine [21].

The development of nAChR agonists began in the early 1990s after the discovery of nicotine’s positive effects. ABT-418, designed by Abbott Labs, was one of the first in a row of nAChR agonists examined as a possible treatment of Alzheimer’s disease, Parkinson’s disease and attention-deficit hyperactivity disorder (ADHD) [27]. Several other antagonist drugs such as varenicline in Champix^® and Chantix^® and nicotine patches are known today to treat tobacco dependence [28]. Drugs like galantamine in Razadyne^®, Nivalin^® are used to treat dementia caused by Alzheimer’s disease. However, its primary mode of action is as an acetylcholine esterase inhibitor. Several other compounds are in clinical trials [29,30].

In mammals, there are 16 different nAChR subunits: nine different α-subunits (α_1–7, α₉ and α₁₀), four β-subunits (β_1–4), as well as γ, δ and ε subunits. Five of these subunits combine to form muscle nAChR subtypes (α₁β₁γδ and α₁β₁δε) which are found at neuromuscular junctions, whereas the rest (α₂–α₁₀, β₂–β₄) assemble in numerous homomeric (having exclusively α-subunits) or heteromeric (having α- and β-subunits) neuronal nAChR subtypes [26]. The assembly of different pentamers forms a complex variety of nAChR subtypes with different pharmacological and biophysical properties. For example, heteromeric receptor subtypes exhibit two distinct subunit stoichiometries of α:β ratios (2:3 or 3:2), each with distinct functional properties that will contribute to synaptic regulation for nicotinic signaling in the mammalian brain [25,31,32]. The diversity increases even further when more than one α or β subunit is included within the same pentamer (for example, α₆α₅β₃ or α₆β₂β₃) [25]. In general, each subunit of a nAChR can be divided into two parts: an extracellular binding domain (ECD) folded into a β-sandwich core, and a transmembrane channel domain (TMD) consisting of four α-helical membrane-spanning segments (M1–M4). Each eukaryotic nAChR subunit also contains an intracellular domain (ICD) consisting of ~100 amino acids defined as the M3-M4 loop (Figure 2) [33]. In each subunit, four flexible loops (loop2, loop7, loop9, and the M2-M3 loop) connect the binding domain to the channel domain and play a crucial role in the coupling of binding site movements to the channel. The binding of neurotransmitter occurs at interfaces between two subunits in the ECD. The M2 helix of each of the subunits forms the ion-conducting channel [19].

Figure 2. Structure and function of the nicotinic acetylcholine receptor. (A) Schematic representation of receptor subunits arranged around a central cation-conducting pore. The ligand binding sites are formed at the interface of two subunits. (B) Illustration of a single nAChR subunit embedded in the membrane. (C) Representation of the protein structure of the pentameric nAChR obtained from T. marmorata (PDB 2BG9) in the plasma membrane. The location and function of the major receptor domains are indicated. A single subunit is highlighted in purple using visual molecular dynamics (VMD). Reproduced from Kabbani et al. (2013) [33], with permission from © 2013 WILEY Periodicals, Inc.

Figure 2. Structure and function of the nicotinic acetylcholine receptor. (A) Schematic representation of receptor subunits arranged around a central cation-conducting pore. The ligand binding sites are formed at the interface of two subunits. (B) Illustration of a single nAChR subunit embedded in the membrane. (C) Representation of the protein structure of the pentameric nAChR obtained from T. marmorata (PDB 2BG9) in the plasma membrane. The location and function of the major receptor domains are indicated. A single subunit is highlighted in purple using visual molecular dynamics (VMD). Reproduced from Kabbani et al. (2013) [33], with permission from © 2013 WILEY Periodicals, Inc.

Investigating how α-conotoxins interact with their targets and which amino acids are important is a challenging research domain. Nevertheless, this information is priceless in the quest for novel and selective therapeutics. In the next sections, we describe the different tools used to determine the mode of action of these α-conotoxins and the important structure-function relationship findings considering α-conotoxins selectively targeting nAChRs.

4. α-Conotoxins and Their Mode of Action—State of the Art

The most important milestones in the determination of the mode of action of α-conotoxins are (i) the discovery of the cryo-electron microscopy structures of the Torpedo nAChR in both a presumed unliganded closed state (4 Å resolution) and liganded open state (6.2 Å resolution) by Unwin and colleagues (2005) [34,35] and (ii) the reporting of the first crystal structure of the acetylcholine binding protein (AChBP) of Lymnaea stagnalis in Nature (2001) by Brejc and colleagues [36] (Figure 3). AChBPs are a class of water-soluble proteins that display significant sequence homology with the ligand-binding domain of α₁ or α₇ nAChRs [37]. The AChBP crystal structure of Brejc and colleagues elegantly reveals the three-dimensional organization of the ACh binding site at 2.7 Å [36]. Since this pioneering work, the structures of AChBP from two other mollusk species and in complex with various ligands have become available. This significantly increased the interest in this protein [38,39,40,41].

Both structures (Torpedo and AChBP) provide excellent tools to model the α-conotoxin/nAChR interactions, but the latter one is currently most used.

Figure 3. AChBP three-dimensional structure (PDB:1I9B) (A) Top view and (B) side view of the Lymnaea stagnalis AChBP. Figures were prepared using the program PyMOL [15].

Figure 3. AChBP three-dimensional structure (PDB:1I9B) (A) Top view and (B) side view of the Lymnaea stagnalis AChBP. Figures were prepared using the program PyMOL [15].

The discovery and description of several X-ray crystal structures of AChBP/α-conotoxin complexes considerably advanced the knowledge of the structural basis for the nAChR subtype selectivity of α-conotoxins. Three conotoxins, ImI [42], PnIA [43] and [A10L]TxIA [7], which have a divergent primary sequence, showed a similar orientation within the ACh binding pocket when they were co-crystallized with AChBP. All of them demonstrated an important contribution of hydrophobic contacts between a conserved proline, several hydrophobic residues of the α-conotoxins and several residues in the aromatic cage of AChBP. Consequently, specific electrostatic interactions and hydrogen bonds formed between the α-conotoxin and the nAChR subunits showed to give rise to different nAChR selectivity profiles [5]. For example, α-conotoxin [A10L]TxIA displays a unique electrostatic pairing between Arg⁵ and AChBP-Asp¹⁹⁵, which is used to achieve the high-affinity binding of [A10L]TxIA (Figure 4) [7]. Moreover, the nAChR subtype selectivity of [A10L]TxIA is thought to arise from a tilt in the orientation of the α-conotoxin structure within the ACh binding pocket.

Figure 4. α-Conotoxin [A10L]TxIA co-crystallized with the Aplysia california AChBP (Ac-AChBP). (A)Top view crystal structure of the Ac-AChBP in complex with [A10L]TxIA (shown in red). (B) Detailed view of the molecular interactions that results in the different backbone orientations of [A10L]TxIA. Reproduced from Dutertre et al., (2007) [7], with permission from © 2007 EMBO.

Figure 4. α-Conotoxin [A10L]TxIA co-crystallized with the Aplysia california AChBP (Ac-AChBP). (A)Top view crystal structure of the Ac-AChBP in complex with [A10L]TxIA (shown in red). (B) Detailed view of the molecular interactions that results in the different backbone orientations of [A10L]TxIA. Reproduced from Dutertre et al., (2007) [7], with permission from © 2007 EMBO.

5. Alpha-Conotoxins and Their Mode of Action

Structure–function activity studies on α-conotoxins appeared in the early 1990s and were mostly alanine-scanning mutagenesis or amino-acid substitution studies. Later, with the crystal structure of the AChBP being available, these investigations were combined with molecular docking studies. In 2001, shortly after the publication of the first AChBP structure, Harel et al. (2001) modeled the interaction of a snake toxin with the nAChR [44]. At this aim, they used NMR data on a complex between α-bungarotoxin, a nicotinic antagonist found in snake venom, and a nAChR peptide mimotope. The complex was then superimposed to the AChBP crystal structure to reveal several important interactions with AChBP loops and side chains. Thanks to the different AChBP structures now available, key interactions as seen in AChBP-ligand co-crystal structures give a clear view of the minimum pharmacophore residues required for binding. The first model of the interaction between an α-conotoxin and nAChRs was described by Dutertre et al. [45], based on docking simulations and distance restrains obtained from mutagenesis data. It has been shown that antagonists such as α-conotoxins make extensive contacts with receptor residues located outside the conserved pocket, whereas agonists appear to make few contacts. Therefore, antagonists allow the design of specific interactions with unique amino acids, as they achieve high subtype selectivity. Here, we describe several studies indicating the interaction of α-conotoxins with neuronal nAChRs (α₇, α₃β₂, α₃β₄, α₄β₂, α₆-containing nAChRs and α₉α₁₀) and muscle subtype nAChRs (α₁β₁γδ and α₁β₁δε). To the best of our knowledge, specific interactions of α-conotoxins with the α₂ subunit have not yet been described.

5.1. Neuronal Subtype nAChRs

5.1.1. α₇ nAChRs Selective α-Conotoxins

One of the neuronal nAChRs, α₇, has received much attention since its discovery [46]. This is due to their distribution in the brain, including regions involved in learning and memory, the hippocampus and the cerebral cortex [47,48,49]. Consequently, α₇ nAChR dysfunctions have been implicated in a variety of severe pathologies such as certain types of epilepsy, myasthenic syndromes, schizophrenia, Parkinson’s and Alzheimer’s diseases [21,50,51]. The binding sites of α₇ nAChRs are formed at the interfaces between identical α₇ subunits in a homopentameric channel. The residues of the α face of the binding site, termed the (+) face, cluster in three well separated regions of the primary sequence, named loops A, B, and C [52] (Figure 5). The stabilization in the AChBP is established based on the vicinal disulfide bonds in loop C, where the α-conotoxin disulfide bond Cys I-III interacts [53].

Figure 5. Structure of one subunit of the α₇ nAChR. The different loops (A–E) and the cys-loop in the extracellular domain as well as the M1–M4 segments in the transmembrane domain and M3–M4 linker in the intracellular domain are indicated. Reproduced from Taly et al. (2006) [54], with permission from © 2006 by The National Academy of Sciences of the USA.

Figure 5. Structure of one subunit of the α₇ nAChR. The different loops (A–E) and the cys-loop in the extracellular domain as well as the M1–M4 segments in the transmembrane domain and M3–M4 linker in the intracellular domain are indicated. Reproduced from Taly et al. (2006) [54], with permission from © 2006 by The National Academy of Sciences of the USA.

One of the first structure–activity relationship studies on α-conotoxins was performed on the closely related conotoxins PnIA and PnIB (C. pennaceus, Figure 6) [55]. The sequences of these toxins differ by only two amino acids, namely Ala versus Leu and Asn versus Ser at position 10 and 11 respectively. Remarkably, PnIA is more potent for α₃β₂ nAChRs, whereas PnIB binds preferentially to α₇ nAChRs. Hogg et al. (1999) [56] and Luo (1999) [57] demonstrated that a Leu for Ala substitution at position 10 makes PnIA a highly selective inhibitor of the α₇ subtype (IC₅₀ of 168 nM). Later, Hogg et al. (2003) [58] showed that changing a single amino acid side-chain at position 10 of PnIA is sufficient to alter the toxin specificity for receptor states in the α₇^L247T mutant. Moreover, the A10L mutation in PnIA changed its properties from antagonistic to agonistic behavior in the α₇^L247T nAChR. The [A10L,D14K]PnIA variant, which behaves similarly to PnIA, was the first conotoxin being co-crystallized in complex with its receptor environment. The toxin was bound to the Ac-AChBP and demonstrated that the protein is mostly buried in the ligand-binding cavity and that no toxin residues are in contact with the AChBP exterior (Figure 7). The N-terminal part is positioned toward the bottom side of AChBP whereas the central helix protrudes into the binding site interior. The C-terminus is located at the top of the binding site with amino acid residues Lys¹⁴-Cys¹⁶ near the outside of the ligand-binding site. The Cys²-Cys⁸ disulfide bond is stacked on the Cys¹⁸⁸-Cys¹⁸⁹ disulfides of the AChBP [43].

Figure 6. Sequence alignment of PnIA, PnIB, ImI, ArIB, LsIA and MrIC. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 6. Sequence alignment of PnIA, PnIB, ImI, ArIB, LsIA and MrIC. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Quiram et al. (2000) demonstrated the existence of a dominant interaction between the α-conotoxin PnIB (C. pennaceus, Figure 6) L¹⁰ and α₇W¹⁴⁹ (located in loop B of the (+) face of the binding site) and weaker interactions between P⁶ and P⁷ of PnIB and α₇Y⁹³ (located in loop A of the (+) face of the binding site) [59]. The authors state the importance of a hydrophobic contribution of residue 10 to the activity towards the receptor. Their overall results placed into close proximity the aromatic side chains W¹⁴⁹, Y⁹³ and Y¹⁵¹ found on the (+) face of the α₇ binding site, and suggested similar interactions for related α-conotoxins. The specificity of conotoxin PnIB for α₇ receptors is due to its rigid scaffold that presents a hydrophobic spiral of side chains to the (+) face of the α₇ binding site.

Figure 7. Representation of two α-conotoxins co-crystallized with Ac-AChBP. Comparison of the Ac-AChBP surface contact area of α-conotoxins [A10L,D14K]PnIA (A) and ImI (B) In the pictures below (C and D), the surface area presentation of both α-conotoxins, (C) [A10L, D14K]PnIA and (D) ImI, protruding in the binding site is shown. Arg⁷ and Trp¹⁰ of ImI are depicted in stick presentation. Reproduced from Ulens et al., (2006) [42], with permission from © 2006 by The National Academy of Sciences of the USA.

Figure 7. Representation of two α-conotoxins co-crystallized with Ac-AChBP. Comparison of the Ac-AChBP surface contact area of α-conotoxins [A10L,D14K]PnIA (A) and ImI (B) In the pictures below (C and D), the surface area presentation of both α-conotoxins, (C) [A10L, D14K]PnIA and (D) ImI, protruding in the binding site is shown. Arg⁷ and Trp¹⁰ of ImI are depicted in stick presentation. Reproduced from Ulens et al., (2006) [42], with permission from © 2006 by The National Academy of Sciences of the USA.

Another α-conotoxin for which structure–activity relationship studies were performed is ImI (C. imperialis, Figure 6). Quiram et al. (1998) identified several determinants (Asp⁵, Pro⁶, Arg⁷, and Trp¹⁰) which influence potency of ImI at the α₇ nAChR [60,61] (Figure 7). The pairwise interactions between ImI and α₇ nAChRs were determined later by thermodynamic mutant cycle analysis [62]. These results revealed a major interaction between Arg⁷ of ImI and α₇Tyr¹⁹⁵, accompanied by smaller contributions between Asp⁵ of ImI and α₇Trp¹⁴⁹, α₇Tyr¹⁵¹ and α₇Gly¹⁵³. Other interactions were found between Trp¹⁰ of ImI and α₇Thr⁷⁷ and α₇Asn¹¹¹. These binding interfaces and conformations were confirmed in co-crystallization experiments of ImI and AChBP [42,63]. Armishaw et al. (2010) used a three-step synthetic combinatorial strategy to study a specific region (i.e., the n-loop AWR) of α-conotoxin ImI to develop novel analogs with improved antagonist properties for the α₇ nAChR. They found that substitutions of Ala⁹ with Nva (norvaline) or Leu residues were optimal for α₇ nAChR activity, whereas the presence of an aromatic residue at the Trp¹⁰ position was observed to be crucial for optimal receptor binding. Substitutions in the Arg¹¹ position had minor effects on antagonistic potency. The most significant increases in antagonist potency were observed for analogs containing the Nva⁹–Dmt¹⁰–His¹¹ (Dmt: 2,6-dimethyltyrosine), Leu⁹–Aph¹⁰–Abu¹¹ (Abu: α-aminobutyric acid), and Nva⁹–Dmt¹⁰–Trp¹¹ combinations which exhibited ~12-, 14- and 10-fold increases in α₇ nAChR inhibition respectively, when compared with wild type ImI.

Whiteaker et al. (2007) [64] synthesized a highly selective α₇ nAChR antagonist by comparing the α-conotoxin ArIB (C. arenatus, Figure 6) with other α-conotoxin sequences. ArIB blocks both α₇ and α₃β₂ nAChRs, but the authors rationally modified the toxin to increase α₇ nAChR selectivity. This structure-function analysis yielded two analogs, [V11L,V16A]ArIB and [V11L,V16D]ArIB, which showed low affinity for α₃β₂ but retained α₇ nAChR activity. An iodinated form of [V11L,V16A]ArIB was later developed as a pharmacological tool with the purpose of facilitating the identification of α₇ nAChRs and enabling the performance of equilibrium binding experiments at α₇ nAChRs [65].

Recently, Inserra et al. (2013) investigated the importance of N-terminal amino acid residues for α-conotoxin LsIA (C. limpusi, Figure 6) binding to different nAChRs. Removing the first amino acid (Ser¹) reduced potency at α₃β₂ and α₇ subtypes by 5- and 2-fold, respectively. Moreover, removing the Ser¹ and Gly² reduced potency by 9- and 4-fold at α₃β₂ and α₇ nAChRs, respectively. They also suggested the importance of the C-terminal chemistry for subtype selectivity [66].

Most α-conotoxins are described as antagonists of the nicotinic acetylcholine receptors, though Jin et al. (2013) recently observed that conotoxin MrIC (C. marmoreus, Figure 6) is an almost full agonist at endogenous human α₇ nAChRs in the presence of PNU, with no activity at endogenous α₃β₂ and α₃β₄ nAChRs in SH-SY5Y cells. However, it should be noted that this agonist activity could not be confirmed on heterologously expressed α₇ nAChRs in Xenopus oocytes. On the contrary, MrIC acted as a simple antagonist at human α₇ nAChRs heterologously expressed in Xenopus oocytes, indicating that significant functional differences of unknown origin exist between neuronal and oocyte expressed α₇ nAChRs. Understanding the structure–activity and mode of nAChR activation by MrIC may influence the improvement of novel α₇ nAChR modulators with potential to treat a range of neurological disorders.

5.1.2. α₃β₂ nAChR Selective α-Conotoxins

The nAChRs including α₃ subunits (α₃*) are found in autonomic ganglia and modulate cardiovascular functions. The α₃* nAChRs expressed by the nociceptive cells in the dorsal root ganglia are likely to modulate pain sensation. In the brain, it is the medial habenula that expresses high α₃* nAChR levels [67]. The habenula is involved in anxiety, fear, and the response to stress. The α₃* nAChRs present in the medial habenula have gained considerable interest because of their potential role in nicotine addiction. When the cholinergic signaling in the medial habenula of mice was blocked, signs of nicotine withdrawal were noticed [68]. Consequently, up- or down-regulation of α₃* nAChR function may influence the dose of nicotine that rodents will self-administer [69,70]. Therefore, strategies to selectively modulate α₃* nAChR function are of substantial interest. The α₃ subunit is structurally closely related to α₆. Consequently, conotoxins that distinguish between α₃* and α₆* nAChRs are rather exceptional.

An α-conotoxin of particular interest is MII (C. magus, Figure 8), which potently blocks α₃β₂- and α₆-containing nAChRs [71]. Harvey et al. (1997) identified specific determinants involved in MII binding on the α₃β₂ nAChR. These residues were Lys¹⁸⁵ and Ile¹⁸⁸ on α₃, and Thr⁵⁹, Val¹⁰⁹, Phe¹¹⁷ and Leu¹¹⁹ on the β₂ subunit [18,72]. With these findings, Dutertre et al. (2005) built an interaction model showing the contribution of the β₂ subunit [18,72].

Figure 8. Sequence alignment of MII, PnIA, PnIB and LvIA. Disulfide bridges are indicated with black lines above the sequences. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. The bold letter in LvIA is an amino acid residue important for α-conotoxin interaction as discussed in this section.

Figure 8. Sequence alignment of MII, PnIA, PnIB and LvIA. Disulfide bridges are indicated with black lines above the sequences. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. The bold letter in LvIA is an amino acid residue important for α-conotoxin interaction as discussed in this section.

As was mentioned in Section 5.1.1., α-conotoxin PnIA is a selective antagonist of α₃β₂, whereas its related sequence, PnIB, binds preferentially α₇ nAChRs [55]. Jin et al. (2008) showed that sequential truncation of the second loop influences potency toward α₃β₂ and significantly alters the structure of PnIA [73]. Concerning the α₃ receptor, Everhart et al. (2003) showed that mutating three specific residues on the α₃ nAChR subunit (Pro¹⁸², Ile¹⁸⁸ and Gly¹⁹⁸) affected the high affinity of PnIA [74]. These structure–activity relationship studies resulted in a molecular docking model for interaction between PnIA and the α₃β₂ nAChR [45]. This model revealed to be consistent with the subsequent co-crystallization structure of the acetylcholine binding protein (AChBP) and a variant of PnIA [43].

Recently, Luo et al. (2014) discovered the first potent α₃β₂-subtype-selective nAChR ligand, named LvIA (C. lividus, Figure 8). Its IC₅₀ value is determined to be 8.67 nM and the amino acid residue Asp¹¹ is believed to play a crucial role in selectivity of LvIA for α₃β₂ versus α₆/α₃β₂β₃ nAChR subtypes. They also performed molecular models of the interactions of LvIA with other nAChR subtypes, which suggested that the specificity of LvIA for α₃β₂ nAChRs may partly arise from electrostatic interactions between Asp¹¹ from LvIA and the receptor. The negatively charged Asp¹¹ showed to be buried in a cluster of charged residues, including Asp¹⁵¹, Lys¹⁵⁴ and Glu¹⁹⁴ of the α₃ subunit and Lys⁷⁸ and Arg⁸⁰ of the β₂ subunit. This cluster of residues forms a globally electropositive environment, which is favorable for an interaction with a negatively charged Asp¹¹. Three other nAChR subtypes, i.e., α₃β₄, α₆β₂β₃ and α₆β₄, display an equivalent cluster with more negative charges, possibly decreasing the affinity for LvIA. Concerning the α₆ subunit, position 154 is occupied by a negatively charged Glu residue, whereas the α₃ subunit has a positively charged Lys residue at this position. Concerning the β₄ subunit, position 78 has a neutral Ile residue, whereas the β₂ subunit has a positively charged Lys residue. The decreased binding of β₄ containing subtypes compared to the α₃β₂ nAChRs may also be explained by the presence of a salt bridge between Lys⁵⁸ and Glu³⁵ of the β₄ subunit which becomes buried when the conotoxin LvIA binds, causing corresponding cost in desolvation energy [75].

5.1.3. α₃β₄ nAChR Selective α-Conotoxins

Because few specific molecular probes toward α₃* nAChRs exist, defining its precise role in normal and pathophysiological conditions is difficult. One particular α-conotoxin, AuIB (Figure 9) from C. aulicus, has been frequently studied. However, due to its lower potency (IC₅₀ of 750 nM), physiological studies are limited.

Figure 9. Sequence alignment of AuIB and TxID. Disulfide bridges are indicated with black lines above the sequences. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 9. Sequence alignment of AuIB and TxID. Disulfide bridges are indicated with black lines above the sequences. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Alpha-conotoxin AuIB is an α4/6-conotoxin and consists of 15 amino acid residues [76]. This peptide is very interesting in several points of view. First, whereas most α-conotoxins inhibiting α₃β₄ nAChRs, also target α₂β₃ nAChR subtypes with similar potency, AuIB exclusively blocks the α₃β₄ nAChR subtype. Second, the non-native ribbon disulfide isomer (I–IV, II–III) of AuIB is more potent than the native globular (I–III, II–IV) AuIB disulfide isomer in rat parasympathetic ganglion neurons. This is in contrast with the general assumption that α-conotoxins with a different disulfide bond connectivity from the native form typically show losses in biological activity [77]. Third, the native globular AuIB was shown to be a non-competitive α₃β₄ antagonist [31], whereas α-conotoxins are generally described as competitive nAChR antagonist [78,79,80]. Remarkably, the AuIB ribbon isomer exhibits subunit stoichiometry-dependent blockade of α₃β₄ nAChRs expressed in oocytes, and unlike globular AuIB, it competitively inhibits α₃β₄ nAChRs [31].

Grishin and coworkers (2013) [81] recently revealed key amino acid residues that affect AuIB-α₃β₄ nAChR interaction. By performing alanine-scanning mutagenesis and molecular dynamics, they found two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, which did not inhibit α₃β₄ nAChRs while [G1A]AuIB only moderately reduced inhibition. Moreover, whereas [F9A]AuIB showed substantially reduced α₃β₄ inhibition, also selectivity for other nAChR subtypes shifted. Further investigation of [F9A]AuIB by NMR and circular dichroism (CD) spectroscopy proved that the peptide retained its native globular structure, whereas the [P6A]AuIB analog structure appeared to be disrupted. Therefore, activity loss of [F9A]AuIB is supposed to be due to loss of specific toxin-receptor residue pairwise contacts. The authors performed homology modeling of the AuIB-α₃β₄ complex which suggested that the N-terminus NH₃⁺ of AuIB forms a salt bridge with the β₄Asp¹⁷² side chain. The G1A mutation introduces a non-polar CH₃ side chain that may weaken this favorable interaction between the peptide N-terminus and β₄Asp¹⁷² side chain. Modeling of the other peptides, [P6A]AuIB and [F9A]AuIB, suggested that Phe⁹ of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. Site-directed mutagenesis of β₄Trp⁵⁹ and β₄Lys⁶¹ residues of loop D which form a putative binding pocket, further confirmed this hypothesis. These experiments suggested that Phe⁹ and Trp⁵⁹ interact via π-π stacking due to the deep insertion of Phe⁹ in the Trp-Leu-Lys (WLK) pocket. When they removed the positively charged Lys⁶¹, the inhibition was reduced, which suggested that this residue likely interacts with Phe⁹ of AuIB and/or stabilizes AuIB-Phe⁹ interaction with β₄Trp⁵⁹. All these findings indicated that Phe⁹ performs a role in the peptide specific interaction with α₃β₄ nAChRs and is needed to maintain selectivity for this particular subtype. Interestingly AuIB and several other α-conotoxins (such as RgIA and Vc1.1 inhibiting α₉α₁₀ and α₉α₁₀/α₇, respectively) exhibit analgesic properties when tested in animal models of pain [13,14] (see also Section 5.1.5).

The only other α4/6 peptide pharmacologically characterized is α-conotoxin TxID (Figure 9), isolated from Conus textile [82]. TxID targets α₃β₄ nAChRs and interestingly it is 60-fold more potent than AuIB, having an IC₅₀ value of 12.5 nM. Nevertheless, TxID also exhibits activity on the closely related α₆/α₃β₄ nAChR subtype (where α₆ and α₃ form a chimeric α subunit) with an IC₅₀ of 94 nM. Surface analysis of both peptides revealed that despite their sequence variation, both have a similar type of surface in terms of biophysical properties on one face and a different surface characteristic on the other face. AuIB has a negatively charged Asp residue at position 14, whereas TxID has a hydrophobic Ile residue in the corresponding surface location. As both peptides fold similarly, the authors state that this difference in surface properties might be the reason for higher selectivity of TxID on α₃β₄ nAChRs.

TxID has a SHP(V) sequence in the first loop, which is also present in other α-conotoxins. This indicates that TxIDs selectivity is probably determined by its unique second loop residues -SAMSPI-. The proline residue in the first loop of TxID is also believed to play a role in the overall conformation as cis-trans isomerism may occur. NMR studies showed that at least two structural isomers are present in TxID.

5.1.4. α₄β₂ nAChR Selective α-Conotoxins

The neuronal nAChRs α₄β₂ are the most abundant nicotinic receptors in the human brain. There, they play special roles concerning the efficiency of synaptic communication by modulating the release of other neurotransmitters [83,84,85]. The α₄β₂ nAChRs are found to play a central role in nicotine addiction and in cognitive processes [83,84,85,86] which makes them potential targets for drugs designed for improved cognition, smoking cessation, the reduction of pain and a variety of neurological disorders such as Alzheimer’s and Parkinson’s disease, depression, and attention deficit disorders [87,88,89].

So far, no conotoxin that selectively targets α₄β₂ nAChRs has been identified, and only a few α-conotoxins (MII [71], GID [90], GIC [91] and AnIB [92], Figure 10) have been shown to block this receptor, although at high nanomolar or micromolar concentrations.

Figure 10. Sequence alignment of GID, TxIA, MII, GIC and AnIB. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. Hydroxyproline residues are indicated as O, γ-carboxyglutamate residues as γ. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 10. Sequence alignment of GID, TxIA, MII, GIC and AnIB. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. Hydroxyproline residues are indicated as O, γ-carboxyglutamate residues as γ. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

The α-conotoxin GID (C. geographus, Figure 10) [90], having a relatively high affinity for the α₄β₂ nAChR subtype, is unusual because it possesses an extended N-terminus of four residues, whereas the N-terminal amino acid residue of α-conotoxins typically is a glycine followed by the first two cysteine residues. Moreover, two post-translational modifications occur before the mature toxin is completed. An entire alanine scan of all non-cysteine residues revealed that most analogs had at least a 10-fold reduced activity at the α₄β₂ subtype, which implies a highly specific interaction of all the amino acids and their charges with the receptor [93]. Recently, Banerjee and colleagues (2014) [94] provided more insight into α-conotoxin GID/nAChR interactions by designing the most α₄β₂ selective conotoxin analogue identified to date, namely [V18N]GID. The authors observed a potential hydrogen bond interaction between the amide functionality of Asn¹⁸ in [V18N]GID and the hydroxyl group of Y¹⁹⁵ of α₄β₂ nAChRs, but not in the α₃β₂ subtype. These interactions appeared to shift the location of the C-loop in the nAChR which might explain the observed selectivity for the α₄β₂ nAChR. Two other GID analogues, [A10S]GID and [V13I]GID, demonstrated moderately improved selectivity toward α₄β₂ over α₃β₂ nAChRs when compared with GID. These observations showed that positions 10, 13 and 18 appear to be major determinants in GID that contribute to selectivity between α₄β₂ and α₃β₂ nAChRs.

Beissner et al. (2012) [95] investigated several α-conotoxins (MII, TxIA and [A10L]TxIA, Figure 10) and found that an arginine residue in position 185 and a proline residue in position 195 of the α₄ subunit prevent efficient α-conotoxin binding. When they replaced these amino acid residues in the α₄ nicotinic receptor subunit by the corresponding residues in the α₃ subunit, they could transfer the low nanomolar potency of α-conotoxin [A10L]TxIA to the α₄β₂ subtype, which is otherwise insensitive to this toxin. They performed docking simulations which revealed an interaction of α₄Arg¹⁸⁵ with the arginine residue in position 5 of α-conotoxin TxIA. The replacement of Arg¹⁸⁵ by isoleucine resulted in a 10-fold (MII) up to at least 1000-fold (TxIA and [A10L]TxIA) enhanced potency of these α-conotoxins at the α₄β₂ receptor subtype. Further, they demonstrated that replacement of α₄Arg¹⁸⁵ by the smaller amino acid Ala or a negatively charged Glu enhanced affinity of the α₄β₂ receptor for [A10L]TxIA. On the contrary, a positively charged Lys did not. Hereupon, they concluded that a positive charge in this position specifically prevents high-affinity binding of most conotoxins to the α₄β₂ nicotinic receptor and thus represents a major determinant for subtype selectivity.

5.1.5. α₆* nAChR Selective α-Conotoxins

The α₆* nAChRs have previously been assumed to be mainly localized to catecholaminergic nuclei of the central nervous system. However, recent data designates that the α₆ subunit is abundantly expressed in visual pathways and is also present in peripheral tissues [96,97,98]. The nAChR α₆ subunit is not widely expressed in the brain, nevertheless it is abundant in midbrain dopaminergic regions which are related to pleasure, reward and mood control [99,100,101,102]. Therefore, Yang et al. (2009) suggested that α₆* nAChRs might play critical roles in nicotinic reward and in the regulation of mood by nicotine [103]. As mentioned earlier, the α₆ subunit is structurally closely related to α₃. Therefore, conotoxins that distinguish between α₆* and α₃* nAChRs are rather exceptional.

Figure 11. Sequence alignment of MII, PIA, PeIA, BuIA and TxIB. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 11. Sequence alignment of MII, PIA, PeIA, BuIA and TxIB. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

As described in Section 5.1.3, conotoxin MII (C. magus, Figure 11) not only blocks α₃β₂- but also α₆-containing nAChRs [71]. McIntosh et al. (2004) designed a series of MII analogs selectively targeting the α₆/α₃β₂β₃ (where α₆ and α₃ form a chimeric α subunit) nAChR combination [104] which were utilized to determine the contribution of α₆-containing nAChRs in dopamine release in the striatum. The most interesting peptide was [H9A,L15A]MII, which the authors put forward as a selective probe for discriminating among numerous nAChR subunit combinations, as this MII analog showed low IC₅₀ value for the α₆/α₃β₂β₃ nAChRs (2.4 nM) and a relatively high IC₅₀ for other nAChRs (α₂β₂, α₂β₄, α₃β₂, α₃β₄, α₄β₂, α₄β₄ and α₇). Another analog, [S4A,E11A,L15A]MII, selectively binds the α₆ versus α₃ subunit by 1000-fold. Three residues were determined to be critical for this selectivity, namely Glu¹⁵², Asp¹⁸⁷ and Thr¹⁹⁸ [105]. Moreover the down-regulation of α₆/α₃β₂β₃ upon long term nicotine exposure could be examined [106,107].

Alpha-conotoxin PIA (C. purpurascens, Figure 11) was the first α-conotoxin shown to discriminate between α₆ versus non-α₆-containing nAChRs. PIA has namely a 75-fold lower IC₅₀ for α₆/α₃β₂β₃ nAChRs compared to α₃β₂ nAChRs. Contrarily, the IC₅₀ for α₄β₂ and α₂β₂ was more than 10 μM. The toxin is believed to bind to determinants on the extracellular portion of the nAChR (i.e., α₆). The remaining α₃ portion of the chimeric α₆/α₃ subunit does not affect peptide binding. When PIA and MII are compared, both toxins have identical spacing of Cys residues, disulfide connectivity, and the SNPV sequence in the first peptide loop. Therefore, the authors state that differences in either the N-terminal or loop 2 sequences account for the differences in selectivity between α₆ and α₃ subunits [108].

The α-conotoxin PeIA (C. purpurascens, Figure 11) is a peptide antagonist blocking several nAChR subtypes, including α₆/α₃β₂β₃ and α₆/α₃β₄ nAChRs, with low nanomolar potency. Hone et al. (2012) [109] systematically mutated PeIA by substituting specific amino acids of PeIA with those of MII. This resulted in the analog [S9H,V10A,E14N]PeIA which potently blocked α₆/α₃β₂β₃ (223 pM) and α₆/α₃β₄ (65nM) nAChRs yielding a >290-fold separation between the two subtypes.

Kim and McIntosh (2012) [110] determined a triad of key residues (Lys¹⁸⁵, Thr¹⁸⁷ and Ile¹⁸⁸) that influence binding of α-conotoxin BuIA (Figure 11) from C. bullatus to the α₆ nAChR subunit. BuIA blocks α₆/α₄β₂β₃ (where α₆ and α₄ form a chimeric α subunit) with an IC₅₀ of 0.43 nM, whereas it blocks α₄β₂ nAChRs with an IC₅₀ of >20 μM. When these amino acids were inserted into the α₄ subunit, there was a 2000-fold increase in toxin potency. Also Thr¹⁹⁸ and Tyr²⁰⁵ were shown to contribute to BuIA potency. Moreover, Thr¹⁹⁸ caused BuIA potency differences between the closely related α₆ and α₃ subunits. Thr¹⁹⁸ appears to be a common denominator in α-conotoxin subtype discrimination of nAChR α-subunits as it was also observed by Azam et al. (2008) [105]. Because Tyr²⁰⁵ is located far from the ligand binding pocket near the boundary with the transmembrane region, the effect on potency by this residue is very likely indirect.

Luo and coworkers (2013) recently reported an α-conotoxin, TxIB (C. textile, Figure 11), which selectively targets α₆/α₃β₂β₃ nAChRs with an IC₅₀ of 28 nM. The toxin has a typical loop 1 Ser-Xaa-Pro motif, but the amino acids “RNKH” in loop 2 are distinct whereupon the authors suggested that the amino acids in loop 2 may be responsible for its selectivity. Other determining factors might be the combination of a smaller hydrophobic patch with flanking positively charged residues of TxIB compared to other conotoxins such as MII, PIA, BuIA, and GIC. As there is a paucity of ligands that can effectively discriminate between α₆β₂ and α₆β₄ nAChRs, the authors believe that the unique selectivity of TxIB will allow probing of nAChR function in tissues where both the α₆* and other nAChR subtypes occur [111].

5.1.6. α₉α₁₀ nAChR Selective α-Conotoxins

The α₉α₁₀ nAChR subtype, being comprised of two α₉ and three α₁₀ subunits [112], is expressed in outer hair cells mediating efferent olivocochlear innervations and in lymphocytes playing a role in immune responses [113,114,115]. Moreover, the α₉α₁₀ nAChR showed to be involved in immune responses, pain [14,116] and in (breast/lung) cancer therapy, functioning as a molecular target [117].

Alpha-conotoxins that target α₉α₁₀ nAChRs are Vc1.1, RgIA and PeIA [78,118,119] (Figure 12). Synthetic Vc1.1 (ACV1) was initially shown to block potently neuronal (α₃, α₅, α₇ and β₄ nAChR subunits) versus muscle nAChRs [11]. Therefore, it was selected for testing in pain models subsequently revealing Vc1.1 as the first α-conotoxin exhibiting efficacy in pain models [13,14]. In 2006, Vincler et al. showed that Vc1.1 is a potent antagonist of α₉α₁₀ nAChRs which potentially contributes to its analgesic effect [12]. Indeed, whereas α₉α₁₀ nAChR-selective antagonists were demonstrated to relieve pain as well, the mechanism of inactivation of N-type calcium channels via G protein-coupled GABA_B receptors was thought to be the principal mechanism of analgesic action [120,121,122,123]. Later, Napier et al. (2012) determined that Vc1.1 fails to block spinal cord N-type calcium channels, raising doubt about this proposed mechanism [124]. Their findings rather suggest that antagonists acting selectively on α₃ subunit containing nAChRs, but not α₄ or α₉α₁₀ subunit-containing nAChRs, may be promising targets in neuropathic pain. ACV1 (Vc1.1) was taken through phase I clinical trials by Metabolic Pharmaceuticals (Melbourne, VIC, Australia), but unfortunately, clinical trials stopped after completion of a phase 2A trial because of potential concerns of efficacy and its reduced affinity at human versus rat α₉α₁₀ nAChRs [5]. Several other α-conotoxins (AuIB and RgIA inhibiting α₃β₄ and α₉α₁₀ nAChRs, respectively) also exhibit analgesic properties when tested in animal models of pain [13,14]. GABA_B receptor-mediated suppression of N-type calcium channels (Ca_V2.2) was here too believed to be the common mechanism of analgesic action [125].

Figure 12. Sequence alignment of Vc1.1, RgIA, PeIA and LvIA. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 12. Sequence alignment of Vc1.1, RgIA, PeIA and LvIA. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Halai et al. (2009) [126] performed scanning mutagenesis studies of Vc1.1 (C. victoriae, Figure 12) revealing the residues Ser⁴ and Asn⁹ as critical determinants for α₉α₁₀ nAChR potency. Mutating Ser⁴ by a more positive residue showed to be more favorable for potency of Vc1.1, whereas mutations to either an Ala or an Asp reduced its activity. If the polar residue Asn⁹ was replaced by a hydrophobic residue (Ala, Leu or Ile), potency of Vc1.1 significantly increased. A molecular docking study of Vc1.1 combined with electrophysiological recordings performed by Yu et al. (2013) [127] showed that position 9 of Vc1.1 had most interactions with non-conserved positions of nAChRs. This amino acid is located in the middle of the short α-helix of Vc1.1. Mutational studies revealed that [N9W]Vc1.1 inhibition of the human α₉α₁₀ nAChR was significantly increased, whereas the potency of [N9F]Vc1.1 to inhibit this receptor was maintained. All these findings strongly suggested that Vc1.1 and its variants preferentially bind the α₁₀α₉ binding site and that the formation of a single hydrogen bond between position 59 of the α₉ subunit and the C-terminal amide of Vc1.1 controls specificity between human and rat receptors [127].

The heteropentamer α₉α₁₀ nAChR displays three potential binding sites located at the α₁₀α₁₀, α₉α₁₀, and the α₁₀α₉ interfaces, where the latter binding site contains more charged residues than the former [127]. Recently, Indurthi et al. (2014) [32] proposed that a fourth possible binding site might exist, i.e., α₉α_9. The α₁₀α₉ interface was previously set up to be the most probable binding site of Vc1.1, which has four charged side chains [127]. By contrast, PeIA (C. pergrandis, Figure 12) has only one charged side chain, Glu¹⁴, and potentially binds to the more hydrophobic α₉α₁₀ pocket. α-conotoxin LvIA (C. lividus, Figure 12), a potent antagonist of α₃β₂ nAChRs (see Section 5.1.2), retains two charged side chains, Glu¹⁴ and Asp¹¹, which are believed to be involved in the toxins’ inaffinity for the α₉α₁₀ nAChR. The latter residue is thought to reduce affinity at the α₉α₁₀ pocket, whereas binding to the α₁₀α₉ pocket was found to be unlikely due to poor shape complementarity [75].

With regard to RgIA (C. regius, Figure 12), the residues Asp⁵, Pro⁶ and Arg⁷ in loop 1 were shown to be critical for both α₉α₁₀ and α₇ nAChR blockade. By contrast, Arg⁹ in loop 2 revealed to be crucial for specific binding to the α₉α₁₀ subtype [128]. In a study from Azam and McIntosh (2012), position 56 of α₉α₁₀ nAChRs was determined to control the species selectivity (rat versus human) of α-conotoxin RgIA. This toxin is 300-fold more potent on rat versus human α₉α₁₀ nAChRs, but it displayed similar activity at the human receptor and at the mutant rα₉α₁₀^T56I nAChR which incorporates the Ile residue present in the human α₉ subunit. Hereupon, they suggested that RgIA preferentially binds the α₁₀α₉ pocket, which contains Thr at position 56 of the α₉ subunit [129].

5.2. Muscle Subtype nAChRs: α₁β₁δε (adult) and α₁β₁γδ (fetal) nAChRs

The muscle subtype nAChRs, α₁β₁δε (adult) and α₁β₁γδ (fetal) nAChRs, are found at the neuromuscular junction. During late gestation, the γ subunit of the neuromuscular nAChR is replaced by the ε subunit in mammalian muscle. Nevertheless, also in adult mammalian tissues, instances of fetal muscle nAChR expression exist. Under normal physiological conditions, expression of the γ subunit occurs in the thymus [130,131] and extraocular muscle fiber [132]. On the contrary, γ subunits are also expressed under pathological conditions such as rhabdomyosarcoma, a pediatric soft-tissue cancer [133,134,135], in denervated muscle [136,137] and muscle tissue associated with various neurogenic and myogenic disorders.

Structurally, each α₁ subunit folds such that the principal binding site directly faces a neighboring subunit, which is either a γ/ε or a δ subunit (Figure 13). The γ subunit is believed to be the one that forms stable contacts being the lone subunit between the two α subunits, while the δ subunit pairs with the β subunit to form stable contacts between the α subunits on the opposite side. As two α₁ subunits are separated by at least one non-α subunit, correct coupling between these subunits is required for cooperative binding of agonists [138]. Agonists of the muscle subtype nAChR initiate channel opening and desensitization by binding to a site on each of these two α₁ subunits, as well as to the γ/(ε) en δ subunits [139]. More specific, Arias and Blanton (2000) established that two adjacent cysteines (at position 192 and 193 according to the sequence number of Torpedo AChR) in the α₁ subunits are involved in the recognition and binding of cholinergic agonists and competitive antagonists [140]. Binding of acetylcholine at both binding sites of the muscle nAChRs induces channel activation [141]. Kinetic studies have shown that the two binding sites differ by 30–100 fold in their affinity for acetylcholine [142,143]. Because this compound must occupy both sites to open the channel, it has been suggested that this difference may be physiologically important in priming the receptor for rapid activation (at the high-affinity site) and in abruptly terminating the response to agonists (low-affinity site). Antagonists that act at either binding site will cause a functional block of the receptor [141]. Agonists and antagonists can specifically distinguish between the α₁γ/(α₁ε) and α₁δ binding sites of the fetal/(adult) muscle acetylcholine receptor because of different contributions by the γ/(ε) and δ subunits where a minimum of four loops in both subunits is required to create the agonist binding site [52].

Figure 13. Schematic representation of the fetal muscle subtype nAChR demonstrating (a) the imbedding in the membrane, showing the synaptic space, the cytosol, as well as the M2 α helix and the gate of the nAChR and (b) the acetylcholine binding sites. In both parts, an indication of the size is included. Reproduced from Khalid (2013) [144], with permission from © 2013 InTech.

Figure 13. Schematic representation of the fetal muscle subtype nAChR demonstrating (a) the imbedding in the membrane, showing the synaptic space, the cytosol, as well as the M2 α helix and the gate of the nAChR and (b) the acetylcholine binding sites. In both parts, an indication of the size is included. Reproduced from Khalid (2013) [144], with permission from © 2013 InTech.

α-Conotoxins which selectively target muscle subtype nAChRs typically have a 3/5 structure [5]. The two most investigated α3/5 conotoxins are α-conotoxin MI (C. magus, Figure 14) and GI (C. geographus, Figure 14). In mammalian muscle nAChRs, both conotoxins showed to preferentially target the α/δ site by 10⁴-fold over the α/γ site [145,146]. Contrarily, in Torpedo nAChRs, their selectivity profile for each site is opposite, where both conotoxins preferentially bind the acetylcholine binding sites located at the α/γ subunit interface versus the α/δ interface [146,147,148]. The explanation for this contradiction was later given by Sine et al. (1995). Using chimeric subunits and site-directed mutagenesis, they identified three determinants at equivalent positions of each subunit that direct selectivity of conotoxin MI for the two binding sites. The amino acid residues Lys³⁴, Ser¹¹¹ and Phe¹⁷² of the γ subunit were found to be responsible for low affinity to the α/γ binding site, whereas the corresponding residues of the δ subunit, Ser³⁶, Tyr¹¹³ and Ile¹⁷⁸, conferred high affinity to the α/δ site. The opposite selectivity earlier experienced in Torpedo AChRs was then explained being caused by a Tyr-cation interaction, because in Torpedo, the second determinant is a Tyr in the high affinity γ subunit, whereas it is an Arg in the low affinity δ subunit [149]. Concerning the ε subunit, residues 106 and 115 of this subunit promote its association with the α subunit, thus affecting efficiency of assembly [150].

Figure 14. Sequence alignment of MII, GI, SrIA, SrIB and EI. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. Hydroxyproline residues are indicated as O, γ-carboxyglutamate residues as γ. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 14. Sequence alignment of MII, GI, SrIA, SrIB and EI. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. Hydroxyproline residues are indicated as O, γ-carboxyglutamate residues as γ. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Loop 1 and loop 2 are labeled below the amino acid sequences. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

A structural binding model of α-conotoxin GI (C. geographus, Figure 14) was established by Ghermann et al. [151]. In conotoxin GI, the high differential selectivity and affinity for the two different acetylcholine binding sites of muscle-type nAChRs, located at the α/δ and α/γ subunit interfaces, is mediated by an α subunit binding face and a selectivity face. The former one is made up of Cys², Asn⁴, Pro⁵, Ala⁶ and Cys⁷ [151] and the latter one is comprised of Arg⁹ and His¹⁰ [149,152,153]. These two faces orient the molecule between the α and δ subunits of the receptor. Another important residue is Tyr¹¹, shown to be vital for binding. It is believed that this amino acid plays a structural role, i.e., assisting in orienting binding epitopes but not directly binding to the receptor.

The α4/7 conotoxin EI (C. ermineus, Figure 14) [154] was the first conotoxin having a 4/7 structure shown to target muscle subtype nAChRs. The toxin selectively binds the α/δ interface of fetal muscle subtype nAChRs. Other α4/7 conotoxins targeting both neuronal (α₄β₂) and the α/δ binding site of fetal muscle subtype nAChRs are SrIA and SrIB (C. spurius, Figure 14). The peptides EI and SrIB both have positive net charges which may contribute to their activity on muscle receptors [155]. SrIA and SrIB have a Tyr at position 4 of the second loop, which is also found in most of the α3/5 conotoxins blocking α₁β₁γδ nAChRs. This Tyr was shown to importantly contribute to the binding of the α₁/δ subunit interface of the muscle nAChRs by α-conotoxin MI [156]. In the first loop, both peptides of C. spurius have Thr position 4 and a Met at position 2 of the second loop, which may also be involved in muscle nAChR binding [157].

Figure 15. Sequence alignment of PrIIIE, PIIIE OIVB and P_IVA. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. Hydroxyproline residues are indicated as O. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Figure 15. Sequence alignment of PrIIIE, PIIIE OIVB and P_IVA. Disulfide bridges are indicated with black lines above the sequences. Dashes are put to make all sequences and intercysteine loops of comparable length. Hydroxyproline residues are indicated as O. The first column indicates the different conotoxins discussed in this section, the second column the name of the Conus species and the third column the according amino acid sequence. Bold letters are amino acid residues important for α-conotoxin interaction as discussed in this section.

Conotoxins that distinguish between the adult and the fetal muscle subtype nAChRs are generally spoken exceptional. Even rarer are the ones selectively targeting the α₁/ε subunit binding site. One example is ψ-conotoxin PrIIIE (C. parius, Figure 15), characterized by Luisma et al. (2008), which shows higher inhibition potency against the adult subtype (IC₅₀ of 245 nM) than the fetal-subtype nAChR (IC₅₀ of 3.24 μM) [158]. The characteristic disulfide connectivity of ψ-conotoxins is typically I–IV; II–V; III–VI compared to I–III; II–IV for α-conotoxins. Moreover, ψ-conotoxins are usually non-competitive nAChR antagonists whereas α-conotoxins are competitive nAChR antagonists [5]. Another ψ-conotoxin PIIIE from (C. purpurascens, Figure 15) shows an IC₅₀ of 7.4 μM on the adult muscle subtype, but no inhibition on the fetal muscle subtype for concentrations up to 10 μM. Although ψ-conotoxin PIIIE functionally inhibits the acetylcholine receptor, it does so by a mechanism other than competitive binding to the acetylcholine ligand site [159]. Teichert et al. (2005) reported αA-conotoxin OIVB from C. obscures (Figure 15), a unique selective inhibitor of the mammalian fetal muscle nAChR (IC₅₀ of 56 nM), whereas affinity for the adult muscle nAChR is more than 1800-fold lower suggesting its preference for the α₁/γ subunit interface [160]. Another αA-conotoxin was investigated by Han et al. (1997) [161] who derived the solution structure of [Pro7,13] αA-conotoxin P_IVA (Figure 15), isolated from C. purpurascens. This competitive nAChR blocker is completely different from the α-conotoxins, in that it has three-disulfide bonds with a I–V, II–III, IV–VI connectivity pattern. From their solution structure, the authors proposed a binding core of residues Tyr⁶, His¹² and Arg¹⁹, which they superimposed on residues Arg⁹, His¹⁰ and Tyr¹¹ of α-conotoxin GI. However, the similar nAChR binding surfaces showed to more likely arise from a combination of the His¹² and Lys¹⁷/Arg¹⁹ side-chains with possible contributions from Asn⁸ and Ala⁹ of αA-P_IVA [151]. According to Groebe et al. (1995), many of the α-conotoxins bind with 10,000-fold higher affinity to the mammalian α₁/δ interface than the α₁/γ interface [146]. Recently, Lebbe et al. (2014) [162] characterized a particular amino acid residue of α-conotoxin Lo1a (C. longurionis, Figure 15) important for discrimination between neuronal and muscle subtype nicotinic acetylcholine receptors. When the C-terminal Asp of Lo1a, which is insensitive for muscle subtype nAChRs, was deleted or replaced by a positive Arg-tail, they observed an adaptation of affinity for the adult muscle subtype α₁β₁δε. IC₅₀ values were as follows: >50 μM (Lo1a), 4.40 μM (Lo1a-ΔD) and 1.47 μM (Lo1a-RRR).

6. Conclusions

This review aims to give an overview of the molecular pharmacology of α-conotoxins that selectively interact with nicotinic acetylcholine receptors. The diverse composition of nAChRs is implicated in the pathophysiology of a number of diseases including epilepsy, schizophrenia, Alzheimer’s disease, Parkinson’s disease, nicotine addiction, etc. Although a lot of effort has already been done which resulted in the indication of crucial determinants for activity on particular nAChRs, a lot of questions still remain. These question marks include on the one hand some mechanisms of actions that are often controversial or still remain to be elucidated and on the other hand the lack of structure-activity data for α-conotoxins selectively targeting α₂ nAChRs. The importance of the characterization of these activity–relationship interactions cannot be neglected, as is illustrated by the number of diseases which are involved. Therefore, enormous challenges are facing future research, but we are hopeful that this will be rewarded, providing a scaffold for selective peptide-engineering which can be used in drug discovery and consequently, disease treatment.