α6 Nicotinic Acetylcholine Receptors: A Pharmacological Challenge with Untapped Therapeutic Promise

α6-containing nicotinic acetylcholine receptors (α6* nAChRs), particularly the α6β2* and α6β4* subtypes, are promising therapeutic targets for neurological disorders such as nicotine addiction, Parkinson’s disease, and pain. However, progress is significantly hampered by two interrelated challenges: difficulties in achieving robust functional expression in vitro and the limited availability of subtype-selective ligands. This review synthesizes the current advances in overcoming these obstacles. We detail the evolution of in vitro models, highlighting the success of engineered constructs such as chimeras and concatamers, targeted mutagenesis, and chaperone co-expression that have facilitated detailed pharmacological investigations. A central focus is on the current ligand landscape, with a critical evaluation of α-conotoxins as premier-yet-limited pharmacological probes available and the persistent challenge in developing selective small-molecule modulators. By integrating current progress in both receptor expression and ligand pharmacology, this review provides a roadmap for future research aimed at developing the specific tools required to fully exploit the therapeutic potential of α6* nAChRs. α6 Nicotinic Acetylcholine Receptors: A Pharmacological Challenge with Untapped Therapeutic Promise Baojian Zhang 1#, Kailin Mao 1#, Linlin Ma 2,3, Yishan Chen 1, Maomao Ren 4, Yang Xiong 4, Dongting Zhangsun 4, Shuai Dong 4*, Sulan Luo 5*, Bingmiao Gao 1* 1 Engineering Research Center of Tropical Medicine Innovation and Transformation, Ministry of Education, Hainan Key Laboratory for Research and Development of Tropical Herbs, School of Pharmacy, Hainan Medical University, Haikou, China; 2 Institute for Biomedicine and Glycomics, Griffith University, Nathan, Queensland, Australia; 3 School of Environment and Science, Griffith University, Nathan, Queensland, Australia; 4 Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou 570228, China; 5 Guangxi Key Laboratory of Special Biomedicine, School of Medicine, Guangxi University, Nanning 530004, China. # These authors contributed equally to this work.

α6-containing nicotinic acetylcholine receptors (α6* nAChRs), particularly the α6β2* and α6β4* subtypes, are promising therapeutic targets for neurological disorders such as nicotine addiction, Parkinson’s disease, and pain. However, progress is significantly hampered by two interrelated challenges: difficulties in achieving robust functional expression in vitro and the limited availability of subtype-selective ligands. This review synthesizes the current advances in overcoming these obstacles. We detail the evolution of in vitro models, highlighting the success of engineered constructs such as chimeras and concatamers, targeted mutagenesis, and chaperone co-expression that have facilitated detailed pharmacological investigations. A central focus is on the current ligand landscape, with a critical evaluation of α-conotoxins as premier-yet-limited pharmacological probes available and the persistent challenge in developing selective small-molecule modulators. By integrating current progress in both receptor expression and ligand pharmacology, this review provides a roadmap for future research aimed at developing the specific tools required to fully exploit the therapeutic potential of α6* nAChRs. Significance Statement α6-containing nicotinic receptors are key targets for Parkinson’s disease and addiction, but progress is hindered by challenges in their functional expression and a lack of selective ligands. This review provides a critical roadmap by detailing the innovative molecular strategies that enable their study in vitro and by comprehensively assessing the current pharmacological toolkit, thereby guiding future efforts to develop targeted therapeutics for these elusive yet vital receptors.

α6-containing nicotinic acetylcholine receptor, Ligand, α-Conotoxin, Heterologous expression, Parkinson’s disease

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels belonging to the Cys-loop superfamily, widely distributed throughout the central and peripheral nervous system as well as non-neuronal tissues 1 . These pentameric receptors mediate fast synaptic transmission upon binding acetylcholine (ACh) or nicotine, playing crucial roles in diverse physiological processes including cognitive function, muscle contraction, inflammation, and autonomic control 2 . The remarkable diversity of nAChRs arises from the combinatorial assembly of various subunits (α1-α10, β1-β4, γ, δ, ε), leading to subtypes with distinct anatomical distributions, physiological functions, and pharmacological properties. Among this diverse family, nAChRs containing the α6 subunit have garnered significant attention due to their unique characteristics and potential therapeutic relevance. The α6 subunit primarily co-assembles with β2* (containing β2 and potentially β3 subunits) and/or β4 subunits, forming receptor subtypes commonly denoted as α6β2* and α6β4* nAChRs 3,4 (Fig. 1A). Unlike many other nAChR subtypes, α6* receptors exhibit a highly restricted expression pattern. They are predominantly localized in midbrain dopaminergic neurons originating from the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc), key regulators of the mesolimbic and nigrostriatal dopamine pathways, respectively 5-7 . Additionally, significant expression is found in retinal ganglion cells and the adrenal medulla 5,6 . This strategic localization firmly implicates α6* nAChRs as critical modulators of dopamine release and neuronal excitability within these circuits 8,9 . Consequently, they are strongly linked to the reinforcing effects of nicotine and the pathophysiology of nicotine addiction 10,11 . Furthermore, their involvement in the nigrostriatal pathway suggests a role in motor control and positions them as potential targets for Parkinson’s disease therapies 12,13 (Fig. 1C). Emerging evidence also points towards their participation in pain perception and potentially other neurological and psychiatric disorders 14 . The fundamental aspects of α6* nAChRs, including their subunit composition, critical brain distribution, key functional roles, and basic ligand interaction principles, are summarized in Fig. 1. [Insert Figure 1 here] Despite their clear importance, elucidating the precise functions and therapeutic potential of α6* nAChRs has been substantially hindered by two major interconnected challenges. Firstly, achieving robust functional expression of specific subtypes in heterologous systems (e.g., Xenopus Oocytes (RRID:SCR_024430) or mammalian cell lines) has proven exceptionally difficult, likely due to complex assembly requirements or the need for specific cellular environments 15 . This has impeded detailed biophysical and pharmacological characterization. Secondly, the development of potent and, crucially, selective pharmacological tools, especially small-molecule ligands suitable for in vivo use, remains a significant bottleneck 4 . While some progress has been made, distinguishing the effects of α6* nAChRs from those mediated by other closely related subtypes (like α4β2* or α3β4*) continues to be a significant challenge. Therefore, this review aims to provide a comprehensive and critical assessment of the current understanding of α6 nAChRs, focusing specifically on the strategies developed to overcome these key challenges. Specifically, we will first survey and evaluate the advancements made in establishing reliable in vitro expression systems for functional α6 nAChRs (Section 2). This section covers the evolution of techniques, including the use of subunit chimeras and concatemeric constructs, highlighting their successes and inherent limitations in mimicking native receptor properties. Subsequently, we delve into the detailed pharmacology of ligands targeting α6 nAChRs (Section 3). This core section systematically discusses orthosteric agonists (Section 3.1), including the endogenous ligand ACh and synthetic agonists, examining their potency and selectivity profiles (or lack thereof), and α-Conotoxin antagonists (Section 3.2.1), focusing on the peptide toxins derived from marine cone snails, which have proven to be invaluable, often highly potent and selective antagonists, serving as essential research tools. Other Antagonists (Section 3.2.2) cover classical non-selective blockers and other synthetic small-molecule antagonists, emphasizing the persistent challenge of achieving subtype selectivity. Section allosteric Modulators (Section 3.3) reviews compounds that bind to sites distinct from the orthosteric binding pocket, offering alternative approaches to modulate receptor function, and discusses the current status of selective modulators for α6*. By integrating the knowledge gained from both expression system development and ligand characterization, this review seeks to consolidate the currently available toolkit for studying α6* nAChRs, underscore the existing gaps in knowledge and technology, and provide insights for future research directions aimed at fully understanding these unique receptors and potentially exploiting them for therapeutic benefit. Models for the Study of α6* nAChRs Investigating the structural characteristics, pharmacological functions, and screening for ligands of α6* nAChRs necessitates robust experimental systems. While native tissues (e.g., slices from brain regions like the striatum, olfactory tubercle, nucleus accumbens, and frontal cortex 12,16,17 ) and genetically modified animals (e.g., gain-of-function mutant rats/mice with alterations like α6L9’S in the TM2 domain 18,19 ) provide valuable insights, in vitro heterologous expression systems are crucial for detailed mechanistic and pharmacological studies. The most commonly employed systems are Xenopus laevis Oocytes (RRID:SCR_024430) 20-22 and mammalian cell lines, such as human embryonic kidney cells (HEK293 (RRID:CVCL_0045)) 23 . These systems, combined with electrophysiological techniques like patch-clamp recording 24,25, allow for the precise measurement of ion currents generated by functionally expressed nAChRs. Although the genes encoding the α6 subunit in different species were cloned in the early 1990s 26,27, reconstituting functional α6* nAChRs in vitro proved challenging 28 . As pentameric complexes assembled from various subunits, functional expression often requires specific subunit combinations. A seminal study in 1996 by Lindstrom and colleagues demonstrated the first functional expression by co-expressing chicken (c) or rat (r) α6 subunits with human (h) β4 subunits in Xenopus Oocytes (RRID:SCR_024430) (forming cα6hβ4 and rα6hβ4). This pivotal work established that α6 is not an orphan subunit and requires a β subunit (β2 or β4) for function 29 . Subsequently, Fucile et al. (1998) reported the expression of functional cα6cβ2, cα6cβ4, and cα6cα3cβ4 receptors in the human BOSC 23 cell line (RRID:CVCL_4401), where ACh-evoked currents were reversibly blocked by methyllycaconitine (MLA), confirming the feasibility of expressing functional α6* nAChRs in mammalian cells in vitro 30 . Further research delineated the subunit requirements for functional human α6* (hα6*) receptors. Kuryatov and colleagues (2000) confirmed that either the β2 or β4 subunit is essential. While α6 alone or paired only with α3, α4, α5, or β3 variants failed to form functional channels (though α6β2 produces binding sites), combinations such as α6β2α3, α6β2α5, α6β4, and α6β4α5 form functional pentameric ion channels capable of generating detectable currents upon ACh stimulation 31 . Despite these advances, achieving robust functional expression of wild-type (WT) hα6β2 receptors in standard systems like Xenopus Oocytes (RRID:SCR_024430) or HEK293/HEK293T (RRID:CVCL_0063) cells remained largely unrealized for years 32,33, although expression in tsA201(RRID:CVCL_2737) cells was reported 34 . Even WT hα6β4 receptors typically express at low levels in both Oocytes (RRID:SCR_024430) and mammalian cells. Table 1 provides a comprehensive summary of heterologous expression strategies for human α6* nAChRs, detailing the challenges encountered and the significant improvements achieved through approaches like auxiliary subunit co-expression or targeted mutagenesis. [Insert Table 1 here] To overcome the notoriously low functional expression levels of α6* nAChRs, particularly human and rodent subtypes, researchers have developed several key strategies beyond optimizing basic conditions (e.g., increasing cRNA amounts 15, extending expression time 35, or incubation with nicotine 34 or menthol 36 ). These include: Co-expression of Auxiliary Subunits (α5 and β3) The α5 and β3 subunits, often considered “accessory,” can incorporate into α6* nAChRs. Co-expression of α5 can form functional receptors like α6β2α5 and α6β4α5, though sometimes resulting in rapid desensitization 31 . While β3 plays a crucial role in vivo 37 and its co-expression can sometimes improve currents in vitro 31,34,35, conflicting data exists, with some studies showing β3 abolishing function in Oocytes (RRID:SCR_024430) 38 . Furthermore, incorporating WT α5 or β3 can alter channel gating and drug sensitivity 39-44 . Consequently, mutant forms, notably α5V9’S and β3V9’S/V13’S, have proven more effective in significantly enhancing the functional expression of α6* receptors 35,38,45 . Chimeras constructed from the highly similar α5 and β3 subunits (e.g., α51-246/β3230-458) also promote expression 45,46 . Whether α5 and β3 directly participate in forming agonist binding sites within α6* receptors remains debated 31,45,47-51 (Fig. 2). [Insert Figure 2 here] Engineered Constructs: Chimeras and Concatamers Given the challenges in expressing functional WT α6* receptors, various engineering strategies were developed. A major breakthrough involved creating chimeric subunits by leveraging the high sequence homology between the poorly expressing α6 subunit and the robustly expressing α3 subunit ( Table 2 ). Kuryatov et al. 31 pioneered this approach by fusing the α6 N-terminal domain to the transmembrane and intracellular domains of α3 (α6/α3 chimeras). They demonstrated both α6/α3β2 and α6/α4β2 formed functional receptors, whereas reverse chimeras (e.g., α3/α6β2) generally did not ( Table 2 & 3 ). The study suggested the α6 N-terminal domain is crucial for subunit assembly and ligand binding, while domains derived from α3 (particularly the large M3-M4 intracellular loop, as confirmed by further dissection studies 15 ) facilitate efficient surface trafficking and functional expression 52 . The resulting α6/α3 chimera, which often retains sensitivity to α-conotoxin MII 31,32, became an invaluable tool. Critically, combining α6/α3 chimeras with specific auxiliary subunits (like β3V9’S or β3V273S) enabled the establishment of stable mammalian cell lines (HEK293T (RRID:CVCL_0063), SH-EP1 (RRID:CVCL_0F47), tsA-201 (RRID:CVCL_2737)) expressing functional α6* receptors 15,33,53,54 . These stable lines, in turn, greatly facilitated high-throughput screening (HTS) for novel α6* ligands using automated patch-clamp technologies 33,55-57 . An alternative engineering approach utilizes concatameric constructs, where subunits are linked head-to-tail by flexible linkers within a single polypeptide chain (e.g., β3-α6-β2-α6-β2) ( Table 2: Part B ). This method ensures a defined subunit stoichiometry and arrangement, and has also successfully produced functional α6* receptors with predictable pharmacology 52,58 (Fig. 3). [Insert Figure 3 here] [Insert Table 2 here] Utilization of Mutant Subunits Site-directed mutagenesis of α6 or its partner subunits has been instrumental. α6* Mutants : The α6L9’S mutation, identified from gain-of-function mice 19, increases nicotine sensitivity and can enhance expression in specific contexts (e.g., with β4β3) but not universally (e.g., with β4 alone) 59 . Recognizing that differences between human and mouse α6 sequences (especially in NTD and M3-M4 loop) impact expression 35,38,60, targeted mutations in hα6 were explored. For instance, mimicking α3 residues in the M1 domain (e.g., α6F223L) improved α6β4 expression 15 . Systematic mutagenesis by Dash and Li identified critical residues within the hα6 NTD (e.g., Asp57, Arg87, Asp92, Ser156, Asn171) essential for function across different α6* combinations, while mutations at other sites (e.g., Arg96, Asp199, Ser233) could enhance function or alter agonist sensitivity 35 . β*Subunit Mutants : As essential partners, modifications to β2 and β4 are also effective. Using a codon-optimized hβ2 significantly boosted current responses of hα6β2β3V9’S receptors 35 . While certain β4 mutations affect α-conotoxin sensitivity 61-64, they may not directly alter α6/α3β4 expression levels 61 . For rodent receptors, specific mβ3 mutants (e.g., mβ3V9’S, mβ3V13’S) markedly increased mα6β4* expression 38,60 . Furthermore, combining the rα6L9’S mutation with engineered β2 subunits (e.g., β2LFM/AAQA, potentially with L9’S) yielded robust currents (>1 μA) for the otherwise difficult-to-express rα6β2 receptor in Oocytes (RRID:SCR_024430) 65 . Co-expression of Protein Chaperones Cellular machinery involved in protein folding, assembly, and trafficking, known as molecular chaperones, are often required for efficient nAChR expression 66,67 . RIC-3 68,69 and NACHO 66,70 are known chaperones influencing nAChR biogenesis. NACHO, in particular, promotes α6β2* assembly but may require additional factors for surface expression 66 . A significant breakthrough came from high-throughput screening, revealing that co-transfection of four accessory proteins—NACHO (assembly), BARP (channel gating enhancement), LAMP5, and SULT2B1 (surface trafficking promotion)—enabled robust functional expression of WT hα6β2β3 receptors 66,70 . BARP and IRE1a have also been implicated in promoting α6β4 assembly and surface expression 71,72 . [Insert Figure 4 here] Specific Approaches for Rodent α6 Receptors In addition to the strategies above, specific tactics have facilitated rodent α6* receptor studies. Chimeric rat α6/α3 subunits yielded highly expressing rα6/α3β2, rα6/α3β2β3, and rα6/α3β4 receptors 73-75 . For challenging WT rodent α6β2 expression, using tagged subunits (e.g., C-terminal FLAG, HA 76, or eGFP/SEP tags 4,77 ) in suitable cell lines (e.g., tsA201 (RRID:CVCL_2737), Neuro-2a (RRID:CVCL_0470)) proved successful. Table 3 provides a comparative summary of functional expression data for rodent (rat and mouse) α6* nAChRs, highlighting both common expression challenges and successful species-specific strategies. [Insert Table 3 here] The development and refinement of these diverse in vitro strategies—including the use of auxiliary subunits, chimeric constructs, targeted mutations, concatameric designs, and molecular chaperones—have largely overcome the challenges associated with the functional expression of α6* nAChRs. The resulting reliable and functional in vitro models are indispensable tools for dissecting detailed structure-function relationships, characterizing the pharmacological profiles, and facilitating the discovery and development of specific ligands targeting this important receptor subtype. The ligands of α6* nAChRs Like other members of the nicotinic acetylcholine receptor (nAChR) family, α6-containing (α6*) receptors are activated by the endogenous neurotransmitter acetylcholine (ACh) at an orthosteric binding site typically located at the interface between an α and a β subunit 78 . The prototypical exogenous agonist, nicotine, the primary psychoactive component of tobacco, also potently activates many α6* receptor subtypes 79 . However, a paramount challenge in the pharmacological study and therapeutic targeting of α6* nAChRs lies in achieving subtype selectivity. The α6 subunit shares significant sequence homology, particularly within the ligand-binding domain, with the α3 subunit, making it inherently difficult to distinguish between α6* and α3* receptors inherently difficult 80 . Furthermore, α6 frequently assembles with the β2 subunit (forming α6β2* receptors), requiring pharmacological distinction from the abundant α4β2* subtype, or with the β4 subunit (forming α6β4* receptors), necessitating pharmacological discrimination from the more prevalent α4β2* and α3β4* subtypes, which often coexist in the same neuronal populations 81 . This lack of readily available, highly selective ligands significantly impedes efforts to precisely delineate the unique physiological and pathophysiological roles of α6* nAChRs using pharmacological tools 57 . Consequently, the development of novel therapeutics aimed specifically at modulating α6* function for potential treatment of conditions such as Parkinson’s disease, nicotine addiction, or pain critically dependends on overcoming this selectivity hurdle to minimize off-target effects 13 . Therefore, the identification, characterization, and optimization of molecules that interact with α6* nAChRs – encompassing orthosteric agonists and antagonists, as well as allosteric modulators – with both high affinity and, crucially, pronounced selectivity over other nAChR subtypes, remains a central focus of research in this field. This section will comprehensively review the diverse chemical landscape of known α6* ligands, discussing key structural classes, their pharmacological profiles, strategies employed to enhance selectivity, and their applications as indispensable research probes or emerging therapeutic candidates. Orthosteric Agonists 1. Classical Agonists The endogenous neurotransmitter acetylcholine (ACh) serves as the natural agonist for all nAChRs, including α6* subtypes 82 . Nicotine, the well-known alkaloid from tobacco (Nicotiana tabacum), acts as a potent exogenous agonist at many nAChR subtypes, including α6β2* and α6β4* receptors, albeit often with varying potencies and efficacies depending on the precise subunit composition 83-85 . Other tobacco alkaloids, such as nornicotine, anabasine, anatabine, and cotinine (a major nicotine metabolite), are generally less potent or efficacious at nAChRs compared to nicotine and ACh (Fig 5A) 85-87 . 2. Key Non-selective and Partially Selective Agonists Several widely studied nAChR agonists exhibit significant activity at α6* receptors but lack subtype selectivity, limiting their utility for specifically probing α6* function in vivo, though they remain valuable research tools (Fig 5A). Epibatidine : Originally isolated from the skin of the Ecuadorian poison frog Epipedobates tricolor, (±)-epibatidine is an exceptionally potent agonist across a broad range of nAChR subtypes, including α6β2* and α6β4* 88-90 . However, its utility is severely hampered by this profound lack of selectivity and significant dose-limiting toxicity 91,92 . Despite these drawbacks, the epibatidine scaffold has been instrumental in the development of high-affinity radioligands for nAChR imaging using positron emission tomography (PET) and single-photon emission computed tomography (SPECT), such as [¹²³I]5-I-A-85380 and [¹⁸F]Nifene ([¹⁸F]F-A-85380). While these tracers bind to α6* receptors, their signal in vivo typically reflects binding to multiple subtypes (predominantly α4β2*) due to the selectivity issue 93-95 . Cytisine and Varenicline : Cytisine, a plant alkaloid, and its derivative varenicline (Chantix(r)/Champix(r)) are well-known partial agonists with high affinity and efficacy primarily at α4β2* nAChRs 96-98 . This profile underlies their successful use as smoking cessation therapeutics 99 . Importantly, both cytisine and varenicline also interact with α6* subtypes. They typically act as agonists or partial agonists at α6β2* receptors, although generally with lower affinity and/or efficacy compared to their action at α4β2* 100-102 . Their activity at α6* receptors might contribute to their overall pharmacological effects or side-effect profiles, but they cannot be considered α6*-selective agents 103 . 3. Synthetic Agonists with Improved Selectivity Recognizing the limitations of non-selective agonists, significant effort has been directed towards synthesizing novel compounds with improved affinity and selectivity for α6* nAChRs over other subtypes, particularly the closely related α3* and the abundant α4β2* and α7 receptors. While achieving high selectivity remains challenging, several chemical classes have yielded promising leads: Pyridyl Ether Derivatives : Compounds based on this scaffold, such as TC-2429 (C22), have shown some preference for activating α6β2* receptors compared to other subtypes like α4β2* and α3β4* in certain assay systems 104,105 . Diazabicyclic Compounds : Certain 3,7-diazabicyclo[3.3.1]nonanes, like AN317 (related to C41), have been reported to exhibit preferential agonist activity at α6* receptors, potentially mediating effects like dopamine release enhancement 14 . (S)-Phenyl-3-heterocyclo-tetrahydroquinoline (PHT) Isomers : Studies on PHT isomers (e.g., C44, C45) demonstrated that subtle stereochemical changes can influence subtype selectivity, with specific isomers displaying enhanced potency or efficacy at α6β2* receptors relative to α3β4* or α4β2* 106-108 . Other Scaffolds : Various other synthetic efforts, exploring structures like isoquinoline derivatives 80,109,110 and modified azabicyclic frameworks 111, have generated agonists with varying degrees of modest α6* preference, often favoring the α6β2* over the α6β4* combination 112 . A particular hurdle has been the development of potent and selective agonists for α6β4* receptors. While many synthetic compounds show activity at α6β2*, targeting α6β4* selectively has proven more difficult. Some azabicyclic amide derivatives, such as A-844606, represent rare examples reported to possess some agonist activity, albeit often partial, with a degree of preference for α6β4* over certain other subtypes like α3β4*, although selectivity over α6β2* or α4β2* may be limited 108 . Overall, while synthetic chemistry has yielded agonists with improved α6* profiles compared to classical ligands, molecules combining high potency, efficacy, and robust selectivity, especially for distinguishing between α6β2* and α6β4* or cleanly separating α6* from α3* and α4β2*, are still largely elusive. [Insert Figure 5 here] Orthosteric Antagonists 1. α-Conotoxins: The Premier Antagonists for α6 nAChRs* Among the diverse arsenal of nAChR ligands, α-conotoxins represent arguably the most valuable class of antagonists for dissecting the pharmacology and function of α6* subtypes. These small, disulfide-rich peptides, derived from the venom of marine cone snails (Conus species), are structurally defined by two disulfide bonds forming characteristic cysteine frameworks (e.g., CysI-CysIII, CysII-CysIV) 113 . Based on the number of amino acids in the intervening loops between their conserved cysteine residues, they are classified into families such as 3/5, 4/3, and 4/7: the first number indicates the number of residues in Loop 1, and the second number indicates the number in Loop 2. Those targeting neuronal nAChRs predominantly belonging to the 4/4, 4/7, and occasionally 4/6 families 113 . Their often exquisite potency and selectivity establish them as the gold standard for studying this challenging receptor family. A select group of α-conotoxins has emerged as indispensable tools, each offering a unique pharmacological profile. α-Ctx MII, one of the most extensively studied, potently inhibits both α6/α3β2* and α6β4* nAChRs, making it a highly selective probe for receptors containing these subunit combinations 114-116 . In contrast, α-Ctx PIA favors α6β4* receptors, providing a means to distinguish it from α6β2* and α3-containing subtypes [119]. Other key toxins include the potent but less selective α-Ctx BuIA, which blocks a broader spectrum of neuronal receptors including α6* 117, and toxins like α-Ctx VnIB and α-Ctx TxIB, which provide additional valuable probes, sometimes with selectivity for α6β2* over α3β2* 118,119 . The binding profiles and selectivity characteristics of these and other key α-conotoxins, such as RegIIA 120,121, are summarized in Table 4. [Insert Table 4 here] The defined structures of α-conotoxins make them highly amenable to rational design, enabling structure-activity relationship (SAR) studies and the engineering of analogues with improved properties. A primary goal has been to enhance selectivity, for instance, to differentiate between the highly homologous α6* and α3* subtypes. The power of this approach is exemplified by the extensive work on α-Ctx PeIA, where targeted amino acid substitutions yielded analogues with dramatically enhanced potency and selectivity for α6β2* receptors (Fig. 6) 122-124 . However, a critical nuance in this field is the species-dependent activity of many toxins. For example, α-Ctx MII shows different potencies on human versus rat α3β2* receptors, and engineered PeIA analogues can display altered selectivity profiles on human versus rodent subtypes 110,125,126 . These findings underscore the necessity of characterizing ligand activity on species-specific receptors to accurately interpret data from animal models. [Insert Figure 6 here] The utility of these toxins as research tools is vast and multifaceted. In vitro, they are routinely used in electrophysiological and binding assays to define the subunit composition and pharmacology of both recombinant and native channels 127,128 . In vivo, their direct administration into specific brain regions allows researchers to probe the pivotal role of α6* receptors in mediating neurotransmitter release, synaptic plasticity, and complex behaviors related to addiction, movement control, and pain processing 129-132 . Furthermore, radiolabeled versions, most notably [¹²⁵I]α-Ctx MII, have been instrumental for the autoradiographic mapping of α6* receptor distribution, complementing immunohistochemical approaches 133,134 . Despite their power as research tools, the therapeutic potential of native α-conotoxins is severely hampered by their peptide nature, which leads to poor oral bioavailability, susceptibility to proteolytic degradation, and limited penetration across the blood-brain barrier (BBB) [138,139]. To bridge the gap from tool to therapeutic, strategies have been actively explored to overcome these limitations. These include chemical modifications like cyclization to enhance stability 135, and the development of sophisticated delivery systems, such as conjugation to virus-like particles (VLPs) 136,137, cell-penetrating peptides (CPPs) 138, or other permeability-enhancing moieties to improve BBB transport 139,140 . Such innovations aim to harness the exquisite selectivity of α-conotoxins for future therapeutic applications targeting α6* receptors in a range of neurological disorders. Other Antagonists In addition to α-conotoxin-derived peptide antagonists, numerous non-peptide compounds have been reported to exhibit antagonistic activity at α6* nAChRs. These compounds display considerable structural diversity, including classical non-selective antagonists, natural products, synthetic small molecules, and novel scaffolds identified through high-throughput screening (HTS). 1. Classical Non-selective Antagonists Mecamylamine and DHβE are two classical non-selective nAChR antagonists widely used to investigate the functional roles of various nAChR subtypes (Fig. 7A). Mecamylamine, originally identified as a ganglionic blocker, effectively inhibits α6-containing receptors (e.g., α6/α3β2β3 and α6/α4β4 receptors, with IC 50 values of 11 μM and 0.5 μM, respectively) 32 . DHβE, a member of the erythrina alkaloid family initially isolated from Erythrina americana, exhibits broad-spectrum antagonism at α4β2, α3β4, α7, and α6-containing receptors (IC 50 values of 1.1 μM and 0.38 μM at α6/α3β2β3 and α6/α4β4 receptors, respectively) 75 . 2. Natural Product-derived Antagonists Several natural products have also demonstrated antagonistic activity at α6* nAChRs, although typically with limited subtype selectivity (Fig. 7A). For example, methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium brownie seeds, inhibits α6/α4β4 receptors with an IC 50 of 260 nM 32 . Catharanthine, an indole alkaloid isolated from Catharanthus species, and its synthetic derivative 18-MC, both effectively block α6/α3β2β3 and α6/α3β4 receptors, although with relatively low potency (IC 50 values of 10.55 μM and 8.78 μM for catharanthine; 3.74 μM and 3.21 μM for 18-MC, respectively) 141 . Additionally, natural products such as atropine and cocaine have also shown non-selective antagonism at α6-containing receptors 38,142-144 . 3. Synthetic Small-molecule Antagonists In recent years, structural modifications and rational design have led to the development of various synthetic small-molecule antagonists, mainly including the following classes (Fig. 7B): (1) Indolizidine-based compounds Indolizidine derivatives such as (-)-237D and related analogues exhibit potent antagonism at α6β2* receptors. For instance, (-)-237D potently inhibits α6β2*-mediated [^3H]-dopamine release with an IC 50 of 0.18 nM 145 . Virtual screening based on the (-)-237D scaffold has also identified several analogues with high affinity, highlighting the potential of this scaffold for drug development 146 . (2) Bispyridinium compounds Bispyridinium compounds, including bPiDDB, bPiDI, and TMPD, display potent antagonistic activity at α6β2* receptors. For example, bPiDDB inhibits nicotine-induced [^3H]-dopamine release with an IC 50 of 5 nM 147, while TMPD effectively blocks nicotine-induced dopamine release with an IC 50 of approximately 500 nM 148 . These compounds have demonstrated therapeutic potential in animal models of nicotine and alcohol addiction 149,150 . (3) Other synthetic antagonists Additionally, structural modifications of the nicotine scaffold, such as N-alkyl nicotinium salts (e.g., N-n-dodecylnicotinium iodide, NDDNI), have shown potent antagonism at α6* receptors (IC 50 = 9 nM) 151 . 4. Novel Antagonists Identified by High-throughput Screening (HTS) High-throughput screening approaches have provided effective strategies for identifying novel α6* receptor antagonists. Mugnaini et al. screened approximately 100,000 compounds using HEK293 cells (RRID:CVCL_0045) expressing α6/α3β2β3V9’S receptors and FLIPRTM calcium imaging, identifying several antagonists with moderate selectivity, including compound 1 with an IC 50 of approximately 126 nM 33 . Bürli and colleagues conducted a larger-scale HTS (approximately 650,000 compounds), discovering the novel brain-penetrant α6* antagonist CVN417 (IC 50 = 86 nM), which demonstrated potential therapeutic effects in animal models of Parkinson’s disease-related motor dysfunction 83,84 . Despite the structural diversity and significant potency of these non-peptide antagonists, most compounds exhibit limited selectivity for α6* receptors, often interacting with other nAChR subtypes. This lack of subtype selectivity restricts their further development as pharmacological tools and clinical drug candidates. Future research should therefore focus on improving subtype selectivity of α6* receptor antagonists to achieve enhanced pharmacological specificity and therapeutic efficacy. [Insert Figure 7 here] Allosteric Modulators Allosteric modulators represent a distinct class of ligands offering alternative strategies for manipulating receptor function. Unlike orthosteric ligands that compete with the endogenous agonist (ACh) for the primary binding site, pure allosteric modulators bind to topographically distinct sites on the receptor complex 78,152,153 (Fig.8A). This binding event induces conformational changes that alter the receptor’s response to orthosteric agonists, without directly activating or blocking the receptor themselves 154 . These modulators are broadly categorized as: Positive Allosteric Modulators (PAMs): Enhance the receptor’s response to an agonist, potentially by increasing agonist affinity, efficacy (maximal response), or channel open probability/duration. Negative Allosteric Modulators (NAMs): Reduce the receptor’s response to an agonist, potentially through opposite mechanisms to PAMs. 1. Positive Allosteric Modulators (PAMs) Significant progress has been made with the identification of AN6001 (Fig.8B), which emerged as a PAM specifically for α6β2* nAChRs 98 . Importantly, AN6001 demonstrated good selectivity against other major nAChR subtypes, including α4β2, α3β4, α7, and muscle-type receptors. In functional assays using HEK293 cells (RRID:CVCL_0045) expressing hα6/α3β2β3V9’S receptors, AN6001 increased both the potency and efficacy of nicotine. Furthermore, it enhanced agonist-induced dopamine (DA) release from striatal synaptosomes and augmented agonist-induced currents and global cellular responses in dopaminergic neurons within the substantia nigra pars compacta (SNc), the primary site of neurodegeneration in Parkinson’s disease. Critically, AN6001 also potentiated the neuroprotective effect of nicotine against 1-methyl-4-phenylpyridinium (MPP + )-induced toxicity in primary dopaminergic neurons 98 . Thus, AN6001 represents a valuable tool for probing α6β2* function and underscores the therapeutic potential of targeting these receptors, particularly in conditions like Parkinson’s disease. Interestingly, low doses of ethanol (EtOH) have also been shown to exhibit PAM effects selectively on α6* nAChRs 155 (Fig.8B) . Concentrations between 0.1-5 mM EtOH significantly enhanced currents mediated by α6* nAChRs, while notably not affecting α3β4, α4β2, or α7 subtypes. This suggests that α6* nAChRs are sensitive targets mediating some effects of low-dose alcohol consumption via a positive allosteric mechanism. Supporting this in vivo relevance, 5 mM EtOH increased the frequency and amplitude of DA transients in mouse nucleus accumbens slices, an effect attenuated by pretreatment with the α6*-preferring antagonist α-conotoxin MII, directly implicating native α6* receptors in ethanol’s modulatory actions 155 . 2. Negative Allosteric Modulators (NAMs) In contrast to the emerging PAMs, selective NAMs specifically targeting α6* receptors remain largely elusive. Galantamine, used clinically as an acetylcholinesterase inhibitor, exhibits complex allosteric modulation at several nAChR subtypes, sometimes acting as a NAM at higher concentrations 156 (Fig.8B) . However, its specific effects and selectivity regarding α6* subtypes in vivo require further clarification. The development of potent and purely selective NAMs for α6* nAChRs remains an unmet need. [Insert Figure 8 here] The discovery of AN6001 and the characterization of ethanol’s effects mark significant advancements in understanding α6* allosteric modulation, which holds theoretical advantages, including the potential for greater subtype selectivity (as allosteric sites may diverge more than orthosteric sites between subtypes) and the ability to fine-tune physiological signaling by modulating, rather than simply switching on or off, the receptor’s response to endogenous ACh 157 . While the discovery of selective allosteric modulators for many nAChR subtypes has progressed, the field still requires a broader range of well-characterized, potent, and selective PAMs and NAMs, particularly those with drug-like properties suitable for in vivo studies and potential therapeutic development 158-160 . Continued exploration of allosteric sites represents a promising avenue for refining our understanding of α6* receptor function and developing novel therapeutic agents with potentially improved specificity and safety profiles . This review highlights the significant potential of α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as targets for therapeutic intervention, particularly in the context of concerning conditions like addiction, Parkinson’s disease, and pain. However, as detailed herein, significant obstacles currently impede progress in this area. Chief among these are the difficulties in achieving reliable functional expression of these receptors in vitro and the pronounced lack of subtype-selective ligands, especially small molecules suitable for drug development. While α-conotoxins have proven invaluable as exquisite pharmacological probes for elucidating α6* receptor function and distribution, their inherent peptide nature presents challenges for broader therapeutic application, leaving a critical gap in the available chemical toolkit. Overcoming these limitations is essential to translating our growing understanding of α6* nAChR pathophysiology into clinical benefit. Future research should prioritize the development of more robust heterologous expression systems and intensify the search for selective non-peptide modulators. Leveraging structural insights and exploring allosteric modulation pathways appear to be promising avenues. Meanwhile, careful application of existing tools in biologically relevant systems will remain important. Ultimately, success in tackling the dual challenges of expression and ligand selectivity will pave the way for novel therapeutic strategies targeting α6* nAChRs for a range of debilitating neurological disorders. Abbreviations nAChRs, Nicotinic acetylcholine receptors; ACh, acetylcholine; AD, Alzheimer’s disease; PD, Parkinson’s Disease; DRG, dorsal root ganglia; MLA, methyllycaconitine; Nic, Nicotine; DhβE, Dihydro-β-erythroidine; Epi, Epibatidine; DA, dopamine; PAMs, positive allosteric modulators. Funding This review was funded by Hainan Provincial International Science and Technology Cooperation Research Project (No. GHYF2025048), Hainan Provincial Key Point Research and Invention Program (No. ZDYF2022SHFZ309), Hainan Provincial Natural Science Foundation of China (No. 823MS031). CRediT authorship contribution statement Baojian Zhang: Conceptualization, Writing – original draft. Kailin Mao: Conceptualization, Writing – original draft. Linlin Ma: Writing – review & editing. Yishan Chen: Writing – review & editing. Maomao Ren: Writing – review & editing. Yang Xiong: Writing – review & editing. Dongting Zhangsun: Writing – review & editing. Shuai Dong: Conceptualization, Writing – review & editing. Sulan Luo: Conceptualization, Writing – review & editing. Bingmiao Gao: Conceptualization, Writing – review & editing. Declaration of Competing Interest The authors declare no conflicts of interest. Data availability No data was used for the research described in the article.

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Dimerization of-Conotoxins as a Strategy to Enhance the Inhibition of the Human 7 and 9 10 Nicotinic Acetylcholine Receptors. 2020; Figure Legends Fig. 1. Key Features of α6-containing Nicotinic Acetylcholine Receptors (α6* nAChRs). (A) Subunit composition of the predominant α6* nAChR subtypes, α6β2β3* and α6β4*. Different subunits (α6, β2, β3, β4) are indicated by distinct colors. The asterisk (*) denotes the possible presence of other auxiliary subunits not explicitly shown and reflects undefined stoichiometry in native receptors. (B) Example mechanism illustrating ligand interaction at α6β2* nAChRs. Top: Nicotine binding activates the receptor, leading to downstream effects such as dopamine (DA) release in target areas like the NAc. Bottom: The presence of a selective antagonist, exemplified here by α-Conotoxin MII (represented as MII), can block nicotine’s effect by preventing receptor activation, thereby inhibiting nicotine-induced DA release. (C) Integrated view of the distribution and functional significance of α6* nAChRs in key dopaminergic pathways. The sagittal brain schematic highlights primary expression sites, including the ventral tegmental area (VTA), substantia nigra (SN), and their projection targets like the nucleus accumbens (NAc) and striatum. Functionally, α6β2* nAChRs within these pathways are pivotal for controlling dopamine (DA) release, thereby driving reward processes, addiction to substances like nicotine and alcohol, and influencing locomotion. Fig. 2. Challenges and the Auxiliary Subunit Strategy for Functional Expression of α6 nAChRs in Heterologous Systems. (A) Illustration of the inherent difficulty in achieving robust functional expression of wild-type (WT) α6β2* or α6β4* nAChRs in standard heterologous systems. Typically, co-expression of only α6 with β2 or β4 subunits in Xenopus Oocytes (RRID:SCR_024430) or mammalian cell lines (e.g., HEK293 cells (RRID:CVCL_0045)) results in inefficient receptor assembly and/or trafficking, leading to low levels of functional receptors at the cell surface (represented by minimal receptor icons on the cell membrane). (B) Depiction of the auxiliary subunit strategy to overcome expression challenges. Co-expression of α6 and β2/β4 subunits along with auxiliary subunits, such as α5 and/or certain variants of β3 (denoted as β3*, which may represent specific mutations like β3V9’S known to enhance function), significantly promotes the proper assembly, forward trafficking, and insertion of functional α6*-containing nAChRs into the plasma membrane. This results in a substantially higher density of functional pentameric receptors (e.g., (α6)₂(β2)₂(α5), (α6)₂(β2)₂(β3*), etc., represented by increased receptor icons) available for electrophysiological and pharmacological characterization. Fig. 3. Strategies for Engineering Construct to Enhance Functional Expression of α6* nAChRs. (A) Chimera Strategy. Illustration depicting the construction of chimeric subunits to overcome poor trafficking of wild-type (WT) α6 subunits while retaining their ligand-binding properties. The N-terminal domain (NTD) of α6, responsible for ligand binding, is shown fused to the transmembrane domains (TM) of a subunit known for robust expression and trafficking (e.g., α3), indicated by a green checkmark. This contrasts with the poor expression/trafficking associated with the WT α6 TM, indicated by a red cross. The resulting α6(NTD)-α3(TM) chimera facilitates the assembly of functional receptors at the cell surface. (B) Concatamer Strategy. Schematic representation of a concatameric construct designed to enforce specific subunit stoichiometry and arrangement. Multiple nAChR subunits (e.g., β3, α6, and β2 as shown) are physically linked in a defined linear sequence within a single polypeptide chain using flexible linkers (‘Linker’). This linear construct, upon expression, folds and assembles into a functional pentameric receptor with a predetermined subunit composition. Figure 4. A Proposed Model for the Maturation and Trafficking of α6β2β3* Nicotinic Acetylcholine Receptors 161 . This model illustrates the proposed sequential roles of key chaperone and associated proteins in the assembly, maturation, and cell-surface expression of functional α6β2β3* receptors. (1) Assembly in the Endoplasmic Reticulum (ER): The chaperone protein NACHO is essential for the initial assembly of individual α6, β2, and β3 subunits into a complete pentameric receptor complex 66 . (2) ER-to-Golgi Transit: During vesicular transport from the ER to the Golgi apparatus, the nascent receptor is thought to transiently associate with SULT2B1 and LAMP5 162 . (3) Golgi Processing and Partner Exchange: Within the Golgi, SULT2B1 and LAMP5 are proposed to dissociate. Subsequently, the receptor associates with BARP, a critical partner for its final maturation and forward trafficking. 162 . (4) Plasma Membrane Expression: The mature α6β2β3* receptor is ultimately inserted into the plasma membrane, where it remains in a stable complex with BARP, ready to function. Fig. 5. Chemical Structures of Representative Orthosteric Agonists for α6-Containing Nicotinic Acetylcholine Receptors. Panel A displays classical and widely studied nAChR agonists: the endogenous ligand acetylcholine (ACh), the prototypical agonist nicotine, the potent non-selective agonist epibatidine, and the α4β2* partial agonist cytisine which also exhibits activity at α6* subtypes. Panel B showcases synthetic agonists developed with efforts towards achieving α6* nAChR selectivity: the pyridyl ether TC-2429, the diazabicyclic compound AN317, the (S)-phenyl-3-heterocyclo-tetrahydroquinoline (PHT) isomer, and the azabicyclic amide A-844606. Fig. 6. Structural Features and Selectivity Engineering of α-Conotoxins Targeting α6* nAChRs. (A) Representative primary and tertiary structure of an α-conotoxin TxIB. Left:. The linear sequence of α-conotoxin TxIB is shown, with cysteine residues (red) forming the characteristic I-III, II-IV disulfide bond framework (blue lines). The numbers indicate the amino acid count within loop 1 (4 residues) and loop 2 (7 residues), defining its 4/7 cysteine framework. The C-terminal ‘#’ indicates amidation. Right: Ribbon diagram of the solution structure of TxIB (PDB ID: 2LZ5), illustrating its compact, disulfide-stabilized fold. The N-terminusand C-terminus are indicated, and disulfide bonds are shown in yellow. (B) Comparative potency profiles of selected α-conotoxins against rat nAChR subtypes. Bar graphs depict the -log(IC 50 ) values for MII, PIA, and the engineered analog PeIA[A7V,S9H,V10A,N11R,E14A] at rα6/α3β2β3 and rα3β2 nAChRs. Higher bars indicate greater potency. (C) Conceptual strategy for engineering α-conotoxin selectivity using PeIA as an example. Specific amino acid substitutions (A7V, S9R, V10A, N11R, E14A; highlighted in purple) via mutagenesis leading to enhanced α6β2* over α3β2* selectivity of the engineered PeIA compared to the parent PeIA, which targets both α6β2* and α3β2*. Fig. 7. Chemical Structures of Representative Non-Peptide Antagonists Targeting α6-Containing Nicotinic Acetylcholine Receptors (α6* nAChRs). (A) Classical Non-selective and Natural Product-derived Antagonists. This panel displays the chemical structures of well-established non-selective nAChR antagonists including Mecamylamine, a classical ganglionic blocker; Dihydro-β-erythroidine (DHβE), a natural product from Erythrina species; Methyllycaconitine (MLA), a potent α7 nAChR antagonist derived from Delphinium alkaloids, which also exhibits activity at some α6* nAChRs; and Catharanthine, a natural Iboga alkaloid that functions as an antagonist at α6*-containing nAChRs, including α6/α3β4* subtypes, and modulates dopamine release. (B) Synthetic Small-Molecule Antagonists. This panel showcases synthetic compounds developed with varying degrees of selectivity for nAChR subtypes. Structures shown include Indolizidine derivatives such as (-)-237D and related analogues; bPiDDB (1,1’-(butane-1,4-diyl)bis(4-((1,4-diazabicyclo[3.2.2]nonan-4-yl)methyl)pyridinium) dibromide), a representative bispyridinium compound; and N-n-dodecylnicotinium iodide (NDDNI), an N-alkyl nicotinium salt derivative. These examples illustrate different chemical scaffolds explored for nAChR antagonism. (C) Novel Antagonists from High-Throughput Screening (HTS). This panel features examples of antagonists identified through high-throughput screening campaigns. Structures include Compound 1 (HTS-C1) identified from a screen targeting α6/α3β2β3V9’S receptors 33, and CVN417, a brain-penetrant compound discovered by Bürli et al 83 . These molecules represent more recent efforts to identify novel α6* nAChR modulators. Figure 8. Mechanisms of Ligand-Receptor Interaction and Chemical Structures of α6* nAChR Allosteric Modulators. (A) Schematic Representation of Receptor Modulation. The panel illustrates two distinct mechanisms of ligand action. The upper panel depicts competitive antagonism, where a competitive antagonist (red sphere) physically occupies the orthosteric binding site, thereby preventing the agonist (green sphere) from binding and activating the receptor. The lower panel demonstrates positive allosteric modulation (PAM), where a PAM (purple sphere) binds to a separate, allosteric site. This binding enhances the receptor’s response to the agonist (bound at the orthosteric site), leading to increased channel opening or potentiation of the ion current. (B) Chemical Structures of Key Allosteric Modulators Targeting α6* nAChRs. Structures are shown for AN6001, a selective positive allosteric modulator (PAM) for α6β2* subtypes; Ethanol (EtOH), identified as a PAM for α6* receptors; and Galantamine, a complex modulator with potential allosteric effects on α6* receptors. Tables Table 1 Summary of Functional Expression Strategies for Human α6-Containing Nicotinic Acetylcholine Receptors (hα6 nAChRs) in Heterologous Systems | α6* Subunit Combination | Strategy/Key Modification | Expression System(s) | Functional Outcome a (Qualitative/Quantitative) | Reference(s) | | Non-functional Combinations | |||| | hα6 alone | WT | Oocytes (RRID:SCR_024430) | Non-functional | 31 | | hα6 + α3/α4/α5 | WT | Oocytes (RRID:SCR_024430) | Non-functional | 31 | | hα6 + β3 | WT | Oocytes (RRID:SCR_024430), HEK293T (RRID:CVCL_0063) | Non-functional | 31,33,38 | | hα6β2-based Receptors | |||| | hα6β2 | WT | Oocytes (RRID:SCR_024430), HEK293T (RRID:CVCL_0063) | Generally Non-functional or Very Low Expression | 15,31,32 | | hα6β2 | WT | tsA201(RRID:CVCL_2737) | Functional | 34 | | hα6β2α3 | WT | Oocytes (RRID:SCR_024430) | Functional | 31 | | hα6β2α5 | WT | Oocytes (RRID:SCR_024430) | Functional | 31 | | hα6β2β3 | WT | Oocytes (RRID:SCR_024430), HEK293T (RRID:CVCL_0063), tsA201(RRID:CVCL_2737) | Functional (but often low/variable levels) | 15,31,34,53 | | hα6β2β3 | Co-expressed β3 V9’S | Oocytes (RRID:SCR_024430) | Enhanced Function (e.g., ~470 nA with 100 µM Nic a ) | 35 | | hα6β2β3 | Co-expressed β3 V273S | HEK293T (RRID:CVCL_0063) | Functional (Enabled stable cell line development) | 33,53 | | hα6β2β3 | Specific α6 NTD mutants (e.g., R96H, D199Y, S233C) b | Oocytes (RRID:SCR_024430) | Enhanced Function (e.g., α6 NTD mutants generate currents of ~1-2 µA with β3V9’S) | 35 | | hα6β2β3 | Co-expression Chaperones c | HEK293 (RRID:CVCL_0045) | Enhanced Function (WT receptor expression significantly boosted) | 66,162 | | hα6β4-based Receptors | |||| | hα6β4 | WT | Oocytes (RRID:SCR_024430), HEK293 (RRID:CVCL_0045), tsA201(RRID:CVCL_2737) | Functional (but typically low/variable expression, e.g., ~300-600 nA ACh 15 ) | 15,31,32,34,59 | | hα6β4α5 | WT | Oocytes (RRID:SCR_024430) | Functional | 31 | | hα6β4β3 | WT | Oocytes (RRID:SCR_024430) | Functional (502-770 nA ACh) | 31,35,38 | | hα6β4β3 | Co-expressed β3 V9’S | Oocytes (RRID:SCR_024430) | Significantly Enhanced Function (e.g., ~2.1 µA ACh) | 38 | | hα6β4β3 | Co-expressed β3 V13’S | Oocytes (RRID:SCR_024430) | Significantly Enhanced Function (e.g., ~0.9 µA ACh) | 38 | | hα6β4 | Co-expressed α5/β3 V9’S Chimera d | Oocytes (RRID:SCR_024430) | Enhanced Function (~225 nA Nicotine) | 45 | | hα6β4 | α6 F223L Mutant | Oocytes (RRID:SCR_024430), tsA201(RRID:CVCL_2737) | Enhanced Function (e.g., ~50-200 nA ACh [Ooc]; EC₅₀ ~9.6 µM ACh [tsA]) | 15 | | hα6β4 | α6 L9’S Mutant | Oocytes (RRID:SCR_024430) | Functional (EC₅₀ ~2.1 µM Nicotine) | 59 | | hα6β4β3 | α6 L9’S Mutant | Oocytes (RRID:SCR_024430) | Enhanced Function (EC₅₀ ~0.89 µM Nicotine) | 59 | | hα6β4 / hα6β4β3 | Specific α6 NTD mutants (e.g., R96H, D199Y, S233C )² | Oocytes (RRID:SCR_024430) | Enhanced Function (e.g., α6 NTD mutants show increased currents vs WT) | 35 | Table 1 Footnotes Functional Outcome: Indicates whether functional ion channels were detected. “Low/Variable” suggests inconsistency or low current amplitudes across studies or conditions. “Enhanced Function” indicates a clear improvement (e.g., larger currents, more reliable expression) compared to the baseline WT combination, often due to the specific modification listed. Quantitative values are illustrative examples from cited studies under specific conditions (agonist concentrations often 100µM - 1mM for ACh, 10-100µM for Nicotine, but vary). Refer to original papers for precise conditions. Abbreviations : WT, Wild-Type; h, human; nAChR, nicotinic acetylcholine receptor; NTD, N-terminal domain; Oocytes, Xenopus laevis Oocytes (RRID:SCR_024430); HEK293 (RRID:CVCL_0045), Human Embryonic Kidney 293 cells; 293T, HEK293T (RRID:CVCL_0063) cells; tsA201(RRID:CVCL_2737), a derivative of HEK293 cells. ACh, acetylcholine; Nic, nicotine. EC₅₀, half maximal effective concentration. µA, microampere; nA, nanoampere. Notes: a The study utilized codon-optimized hα6 (hα6o) 35 . b Refers to enhancing point mutations identified in the study 35, such as R96H and D199Y. c Refers to co-expression with a chaperone cocktail including NACHO, BARP, LAMP5, and SULT2B1 66,162 . d Refers to the α5(1-246)/β3(230-458)V9’S chimera 45 . Table 2 Functional Expression of Chimeric and Concatenated α6-Containing Nicotinic Acetylcholine Receptors (α6* nAChRs) | Construct Description | Partner Subunit(s) | Auxiliary / Mutant Subunit(s) | Expression System(s) | Functional Outcome / Key Finding | Reference(s) | | Part A: Chimeric Constructs | ||||| | α6 N-Term / α4 C-Term Chimeras | ||||| | α6/α4 a | β2 | — | Oocytes (RRID:SCR_024430) | Functional (ACh ~1 µA, EC₅₀ ~56 µM; Nic EC₅₀ ~3.2 µM) | 31 | | α6/α4 a | β4 | — | Oocytes (RRID:SCR_024430), HEK293 (RRID:CVCL_0045) | Functional (Ooc: ACh ~1-18 µA, EC₅₀ ~37 µM; HEK293 (RRID:CVCL_0045): ACh EC₅₀ ~11 µM) | 32 | | α6/α4 | β2 | + β3 | HEK293 (RRID:CVCL_0045) | Non-functional | 53 | | α6/α4 | β2 | + β3 V9’S | HEK293 (RRID:CVCL_0045) | Functional (Enabled HTS assay; 26% responders >120 pA) | 53 | | α6 N-Term / α3 C-Term Chimeras (Simple Swaps) | ||||| | α6/α3 a (e.g., α61-207/α3208-446) | β2 | — | Oocytes (RRID:SCR_024430) | Functional (ACh ~1 µA, EC₅₀ ~14 µM); Blocked by α-Ctx PIA | 31,73 | | α6/α3 a | β2 | + α5 | Oocytes (RRID:SCR_024430) | Functional (~20 nA Nic) | 45 | | α6/α3 a | β2 | + α5 V9’S | Oocytes (RRID:SCR_024430) | Enhanced Function (~250 nA Nic) | 45 | | α6/α3 a | β2 | + β3 | Oocytes (RRID:SCR_024430), HEK293 (RRID:CVCL_0045) | Functional; Blocked by α-Ctx PIA; Enabled HTS assay (16% responders >120 pA) | 53,73 | | α6/α3 a | β2 | + β3 V9’S | HEK293 (RRID:CVCL_0045) | Enhanced Function (Enabled HTS assay; 57% responders >120 pA) | 53 | | α6/α3 a | β2 | + α5/β3 V9’S chimera | Oocytes (RRID:SCR_024430) | Enhanced Function (~250 nA Nicotine) | 45 | | α6/α3 a (e.g., α61-237/α3239-505) | β2 | + β3 V273S | HEK293T (RRID:CVCL_0063) | Functional; Blocked by α-Ctx MII (Used for establishing a stable cell line) | 33 | | α6/α3 | β2 | + β3 / β3 V273S | HEK293 (RRID:CVCL_0045), SH-EP1 (RRID:CVCL_0F47) | Functional (Basis for multiple HTS campaigns) | 55,56 | | α6/α3 a or α6/α3 b | β4 | — | Oocytes (RRID:SCR_024430), tsA201(RRID:CVCL_2737) | Functional (ACh 3-4 µA [Ooc]; EC₅₀ ~14 µM [tsA]); Blocked by α-Ctx MII & PIA | 15,31,73 | | α6 N-Term / α3 C-Term Chimeras (Loop Swaps)* c | ||||| | α6(1-208)/α3(209-437)/α6(426-464) | β4 | — | tsA201(RRID:CVCL_2737) | Functional (ACh EC₅₀ ~11 µM) | 15 | | α6(1-305)/α3(306-437)/α6(426-464) | β4 | — | tsA201(RRID:CVCL_2737) | Functional (ACh EC₅₀ ~2.4 µM) - Key functional construct | 15 | | α6(1-400)/α3(412-437)/α6(426-464) | β4 | — | tsA201(RRID:CVCL_2737) | Functional (ACh EC₅₀ ~4.7 µM) - Key functional construct | 15 | | α6(1-305)/α3(loop)/α6 + F223L | β4 | α6 F223L mutation | tsA201(RRID:CVCL_2737) | Enhanced Function (ACh EC₅₀ ~0.5 µM) | 163 | | α6(1-400)/α3(loop)/α6 + F223L | β4 | α6 F223L mutation | tsA201(RRID:CVCL_2737) | Enhanced Function (ACh EC₅₀ ~2.0 µM) | 163 | | α6(1-425)/α3(438-474) | β4 | — | tsA201(RRID:CVCL_2737) | Non-functional (Indicates importance of specific α3 loop region) | 15 | | α6(1-343)/α3(344-382)/α6(372-464) | β4 | — | tsA201(RRID:CVCL_2737) | Non-functional (Indicates importance of specific α3 loop region) | 15 | | Reverse Chimeras (α3/α6 or α4/α6) | ||||| | α3/α6 d | β2 | — | Oocytes (RRID:SCR_024430) | Non-functional | 31 | | α3/α6 d | β4 | — | Oocytes (RRID:SCR_024430) | Very Low / Non-functional | 31 | | α4/α6 e | β2 | — | Oocytes (RRID:SCR_024430) | Non-functional | 31 | | Part B: Concatenated Constructs | ||||| | β3-α6-β2-α6-β2 | — | — | Oocytes (RRID:SCR_024430) | Functional (~100 nA with 1 µM ACh) | 58 | | β3-α4-β2-α6-β2 | — | — | Oocytes (RRID:SCR_024430) | Functional (~400 nA with 1 µM ACh) | 58 | | β3-α6-β2-α4-β2 | — | — | Oocytes (RRID:SCR_024430) | Functional (~800 nA with 1 µM ACh) | 58 | Table 2 Footnotes Functional Outcome: Indicates detection of functional ion channels. Quantitative values (currents, EC₅₀) are representative examples under specific conditions; refer to original papers. “Enabled HTS assay” indicates the construct was successfully used for drug screening. “Blocked by…” indicates sensitivity to specific antagonists. Abbreviations : WT, Wild-Type; N-Term, N-terminal domain; C-Term, C-terminal and transmembrane domains; Oocytes, Xenopus laevis Oocytes (RRID:SCR_024430); HEK293 (RRID:CVCL_0045), Human Embryonic Kidney 293 cells; 293T, HEK293T (RRID:CVCL_0063) cells; tsA201(RRID:CVCL_2737), a derivative of HEK293 cells; SH-EP1 (RRID:CVCL_0F47), Human neuroblastoma cell line. ACh, acetylcholine; Nic, nicotine; α-Ctx, α-conotoxin; MII, α-Ctx MII; PIA, α-Ctx PIA. EC₅₀, half maximal effective concentration; HTS, High-Throughput Screening. nA, nanoampere; µA, microampere; pA, picoampere. Notes: a Refers to chimeras swapping the N-terminal extracellular domain of α6 with the transmembrane and C-terminal domains of α4 or α3 (e.g., α6 residues ~1-207 fused to α4/α3 residues ~208-end). Specific constructs may vary slightly between references. b Refers to chimera α6cys1-208/α3209-474 from Ref 15 . c Refers to chimeras where specific segments, particularly within the large M3-M4 intracellular loop, were swapped between α6 and α3. The notation indicates the origin of different segments (e.g., α6(1-208)/α3(209-437)/α6(426-464) means α6 N-term, α3 loop segment, α6 C-term). Only key examples illustrating functional rescue or failure are shown. d Refers to the reverse chimera (α3 N-Term / α6 C-Term), e.g., α3(1-207)/α6(208-464). e Refers to the reverse chimera (α4 N-Term / α6 C-Term), e.g., α4(1-207)/α6(208-464). Table 3 Summary of Functional Expression of Rodent α6-Containing Nicotinic Acetylcholine Receptors (rα6 nAChRs) | Species | Receptor Combination / Modification | Expression System | Functional Outcome / Key Finding | Reference(s) | | α6β2-based Receptors | |||| | Rat | rα6β2 (WT) | Oocytes (RRID:SCR_024430) | Non-functional | 73 | | Mouse | mα6β2 (WT) | Oocytes (RRID:SCR_024430) | Non-functional | 38 | | Mouse | mα6β2 + β3 (WT) | Oocytes (RRID:SCR_024430) | Non-functional | 38 | | Mouse | mα6β2 + β3 (V9’S mutant) | Oocytes (RRID:SCR_024430) | Non-functional / Very Low | 38,60 | | Rat | rα6/α3 + β2 | Oocytes (RRID:SCR_024430) | Functional (Blocked by α-Ctx PIA) a | 73 | | Rat | rα6/α3 + β2β3 | Oocytes (RRID:SCR_024430) | Functional (Blocked by α-Ctx PIA) a | 73,164 | | Rat | rα6/α4 + β2β3 | Oocytes (RRID:SCR_024430) | Functional (ACh EC₅₀ ~20 µM) | 74 | | Rat | rα4/α6 + β2β3 | Oocytes (RRID:SCR_024430) | Non-functional | 74 | | Rat | rα6 L9’S + β2 (WT) | Oocytes (RRID:SCR_024430) | Non-functional | 65 | | Rat | rα6 L9’S + β2 (LFM/AAQA mutant) b | Oocytes (RRID:SCR_024430) | Functional (Moderate current, ≤250 nA) | 65 | | Rat | rα6 L9’S + β2 (L9’S + LFM/AAQA mutant)² | Oocytes (RRID:SCR_024430) | Enhanced Function (Robust current, >1 µA) | 65 | | Rat | rα6-FLAG + β2-HA | tsA201(RRID:CVCL_2737) | Functional (Low current, ~166 pA; ACh EC₅₀ ~46 µM) | 76 | | Mouse | mα6-eGFP + β2 | Neuro-2a | Functional (Low current, ~54 pA; ACh) | 4,77 | | Mouse | mα6-eGFP + β2β3 | Neuro-2a | Functional (ACh) | 77 | | Mouse | mα6-SEP + β2β3 | Neuro-2a | Functional (ACh) | 77 | | α6β4-based Receptors | |||| | Rat | rα6β4 (WT) | Oocytes (RRID:SCR_024430) | Functional (Blocked by α-Ctx) a | 73 | | Mouse | mα6β4 (WT) | Oocytes (RRID:SCR_024430) | Functional (Low current, <100 nA Nicotine) | 38,42,60 | | Mouse | mα6-SEP + β4 | Neuro-2a | Functional (ACh) | 77 | | Rat | rα6β4β3 (WT) | Oocytes (RRID:SCR_024430) | Functional (Blocked by α-Ctx) a | 74 | | Mouse | mα6β4β3 (WT) | Oocytes (RRID:SCR_024430) | Functional (Low current reported, ~85 nA Nicotine 60 ; No function 38 ) | 38,60 | | Mouse | mα6-SEP + β4β3 | Neuro-2a | Functional (ACh) | 77 | | Rat | rα6/α3 + β4 | Oocytes (RRID:SCR_024430) | Functional (Blocked by α-Ctx MII & PIA) a | 61,73 | | Rat | rα4/α6 + β4 | Oocytes (RRID:SCR_024430) | Functional (ACh EC₅₀ ~6.3 µM) | 74 | | Rat | rα6 V177I /α3 + β4 | Oocytes (RRID:SCR_024430) | Functional (Altered α-Ctx sensitivity study) | 61 | | Mouse | mα6 L9’S + β4 (WT) | Oocytes (RRID:SCR_024430) | Functional (Low current ~29 nA Nicotine) | 42 | | Mouse | mα6 L9’S + β4β3 (WT) | Oocytes (RRID:SCR_024430) | Functional (Low current ~26 nA Nicotine) | 42 | | Mouse | mα6β4 + β3 (V9’S mutant) | Oocytes (RRID:SCR_024430) | Significantly Enhanced Function (~2.5 µA; Nicotine EC₅₀ ~0.07 µM) | 38 | | Mouse | mα6β4 + β3 (V13’S mutant) | Oocytes (RRID:SCR_024430) | Significantly Enhanced Function (~3 µA; Nicotine EC₅₀ ~0.26 µM) | 38 | | Mouse | mα6 V13’S + β4β3 (WT) | Oocytes (RRID:SCR_024430) | Enhanced Function (~800 nA; Nicotine EC₅₀ ~1.2 µM) | 42 | | Mouse | mα6β4 + β3 (S144N/S148V mutant) | Oocytes (RRID:SCR_024430) | Enhanced Function (~266 nA; Nicotine EC₅₀ ~10 µM) | 60 | | Mouse | mα6β4 + β3 (E221D mutant) | Oocytes (RRID:SCR_024430) | Enhanced Function (~199 nA; Nicotine EC₅₀ ~4 µM) | 60 | | Rat | rα6/α3 + β4 (Various point mutants) c | Oocytes (RRID:SCR_024430) | Functional (Used to probe α-Ctx MII binding/sensitivity) | 61,165 | Table 3 Footnotes Functional Outcome: Indicates detection of functional channels. Specific blockers, agonist used (ACh or Nicotine), current levels, or EC₅₀ values are noted where reported/relevant. “Blocked by α-Ctx” implies sensitivity confirmed. “Low current” generally implies 1 µA, but these are rough guides. Abbreviations : r, rat; m, mouse; WT, Wild-Type; Oocytes, Xenopus laevis Oocytes (RRID:SCR_024430); tsA201(RRID:CVCL_2737), a derivative of HEK293 cells; Neuro-2a, Mouse neuroblastoma cell line; FLAG/HA, epitope tags; eGFP, enhanced Green Fluorescent Protein; SEP, Super Ecliptic pHluorin; α-Ctx, α-conotoxin; MII, α-CtxMII; PIA, α-CtxPIA; ACh, acetylcholine; Nic, nicotine; EC₅₀, half maximal effective concentration; nA, nanoampere; µA, microampere; pA, picoampere. Notes: a References often confirm function via block by relevant α-conotoxins (e.g., MII, PIA) sensitive to α6*. b Refers to engineered rat β2 subunits with mutations (LFM/AAQA and L9’S) designed to improve expression/function when paired with rα6 L9’S 65 . c Refers to various β4 point mutants (e.g., V110L, I118V, Q119L 61 ; D168A, E34M, K57T 165 ) tested with rα6/α3 chimera primarily for α-Ctx sensitivity studies. Table 4 Pharmacological Profile of Key α-Conotoxins Targeting α6* nAChRs | α-Conotoxin | Sequence | Family | α6β2* IC₅₀ (nM) | α6β4* IC₅₀ (nM) | α3β2* IC₅₀ (nM) | α3β4* IC₅₀ (nM) | α7* IC₅₀ (nM) | α4β2* IC₅₀ (nM) | Ref. | Notes | | MII | GCCSNPVCHLEHSNLC | 4/7 | 0.39 (r) | 1.5 (h) / 32 (r) | ~4 (r) | 1.6 µM (r) | >1000 | >3000 | 32,107,108 | Potent α6/α3β2/β4 blocker; good selectivity vs α7/α4β2 | | PIA | RDPCCSNPVCTVHNPQIC | 4/7 | 0.7-1.7 (h/r) | 13 (h) / 33 (r) | 74 (r) | 518 (r) | >10000 | >10000 | 73 | Highly potent on α6β2*; good selectivity vs α3 subtypes | | BuIA | GCCSTPPCAVLYC* a | 4/4 | 0.26 (r) | 1.5 (r) | 6-8 (h/r) | 28 (r) | 272 (r) | >10000 | 95,97 | Broad spectrum; high potency, low selectivity | | VnIB | GGCCSHPVCYTKNPNCG | 4/6 | >4000 (r) | 5.3 (h) / 18 (r) | >10000 (r) | 320 (r) | >10000 | >10000 | 98 | Highly selective for α6β4* over other subtypes | | TxIB | GCCSDPPCRNKHPDLC | 4/7 | 28-42 (r) / 291 (h) | 402 (h) / >10000 (r) | >10000 (r) | >10000 (r) | >10000 | ND | 61,100 | Potent α6β2* blocker with strong species differences | | PeIA | GCCSHPACSVNHPELC | 4/7 | 11-20 (h/r) | 10 (h) / 148 (r) | ~20 (r) | 1500 (r) | >1800 (r) | >10000 | 109,110,159,166 | Parent peptide for engineering; potent on α9α10 | | [PeIA Analogue] b | GCCSHPVCHARHPALC | 4/7 | 2.2 (r) | 44 (r) | ND | >10000 | >10000 | >10000 | 80 | Engineered for exceptional α6β2* potency and selectivity | | RegIIA | GCCSHPACNVNNPHIC | 4/7 | ND | 147 (r) | 33 (r) | 46-97 (h/r) | 41 (r)/150 (h) | >1000 | 123,158 | Broad-spectrum antagonist for α6*, α3*, and α7* subtypes. | Table 4 Footnotes IC₅₀ Values: Data are indicative and represent approximate potencies often derived from blocking ACh-induced currents in Xenopus oocytes or cell lines expressing the specified receptor subtypes. Species are indicated where available (h: human, r: rat, m: mouse). “Potent” generally implies low nM IC₅₀. ND: Not Determined or not reported in key references. Values from α6/α3βX combinations often reflect the potency at α6βX*. Notes: a * indicates C-terminal amidation. b [PeIA Analogue]: Represents PeIA[A7V, S9H, V10A, N11R, E14A], an example of engineered selectivity. Information & Authors Information Version history Peer review timeline Published European Journal of Medicinal Chemistry Version of Record1 Jan 2026Published Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Metrics & Citations Metrics Article Usage 490views 166downloads Citations Download citation Baojian Zhang, Kailin Mao, Linlin Ma, et al. α6 Nicotinic Acetylcholine Receptors: A Pharmacological Challenge with Untapped Therapeutic Promise. Authorea. 20 August 2025. 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