“Unraveling the role of CHRNA6, the neuronal α6 nicotinic acetylcholine receptor subunit”

Abstract

The increased prevalence of electronic cigarettes, particularly among adolescents, has escalated concerns about nicotine addiction. Nicotine, a potent psychostimulant found in tobacco products, exerts its effects by interacting with nicotinic acetylcholine receptors (nAChRs) in the brain. Recent findings in both pre-clinical and clinical studies have enhanced our understanding of nAChRs, overcoming limitations of pharmacological tools that previously hindered their investigation. Of particular interest is the α6 subunit, whose expression peaks during adolescence, a critical period of brain development often marked by the initiation of substance use. Pre-clinical studies have linked α6-containing nAChRs (α6*nAChRs) to nicotine-induced locomotion, dopamine release, and self-administration behavior. Furthermore, clinical studies suggest an association between the α6 subunit and increased smoking behavior in humans. Specifically, a single nucleotide polymorphism in the 3’ untranslated region of the CHRNA6 gene that encodes for this subunit is linked to smoking behavior and other substance use. A comprehensive understanding of this subunit’s role in addiction is of high importance. This review aims to consolidate current knowledge regarding the α6 subunit’s functions and implications in addiction and other disorders, with the hope of paving the way for future research and the development of targeted therapies to address this pressing public health concern.

Graphical Abstract

Introduction

Nicotine use remains one of the most preventable threats to public health and leading cause of premature death in the United States (USDHHS, 2020). Nicotine is a potent, addictive psychostimulant found in tobacco products. An estimated 46 million adults in the United States reported daily use of any tobacco product during 2021, including electronic cigarettes (e-cigarettes) (Cornelius et al., 2023). Concerningly, an estimated 6.21 million middle and high school students reported trying any kind of tobacco product in the United States in 2022 (Birdsey et al., 2023). Early use greatly increases the likelihood of developing dependence or escalation to more harmful substances (Yuan et al., 2015; Ren & Lotfipour, 2019; Leslie et al., 2020). Smoking and smoking-related diseases adversely impact individual healthcare and imposes substantial economic burden (Xu et al., 2021). Such prevalence in tobacco use and cessation challenges make it necessary to gain a comprehensive understanding of nicotine’s impact on the brain and consequent behaviors.

Nicotinic acetylcholine receptors (nAChRs) serve as key regulators within the brain and their role in nicotine dependence has been well established over decades (Champtiaux et al., 2003; Azam et al., 2007; Changeux, 2010; Zoli et al., 2015; Moen & Lee, 2021). Expression patterns and binding properties of nAChR subunits vary across brain regions but follow distinct developmental events in the cholinergic system (Azam et al., 2007; Dwyer et al., 2009; Thorpe et al., 2020). The subunit configuration of these receptors regulates pharmacological properties such as agonist affinity/efficacy, desensitization, downstream signaling, and is heavily implicated in presynaptic neurotransmitter release (Changeux, 2010; Engle et al., 2013). The α6 nAChR subunit, highly expressed in dopamine neurons within the ventral tegmental area (VTA), has been found in preclinical studies to be crucial for nicotine-induced locomotion, dopamine release, and nicotine self-administration (Pons et al., 2008; Drenan et al., 2010; Gotti et al., 2010; Sanjakdar et al., 2015). Genome-wide association studies have also associated the α6 subunit gene, CHRNA6, with nicotine and other substance use (Dawes et al., 2009; Hoft et al., 2009 a; Lotfipour et al., 2010; Cannon et al., 2014; Pugach et al., 2017). The goal of this review is to evaluate advances in the study of the α6 subunit, its role in substance use disorders and other disease, evaluate genetic influences on behavior, and discuss potential implications for the development of future therapeutic treatments.

1. Structure and distribution of α6-containing neuronal nAChRs

1.1. Structure of neuronal nAChRs

Neuronal nAChRs are ligand-gated ion channels found in the central and peripheral nervous systems (Gotti et al., 2009). The endogenous neurotransmitter acetylcholine (ACh) and exogenous ligands such as nicotine bind to these pentameric receptors, which are composed of five subunits (Dani, 2015). In vertebrates, nAChRs exhibit diverse subunit compositions, with nine ɑ (α2-α10) and three β subunits (β2-β4) identified (Gotti & Clementi, 2004; Corringer et al., 2006; Hurst, 2013). Each of these subunits is made up of a long extracellular N-terminus, that is involved in ligand binding, four transmembrane domains (M1–M4), an intracellular cytoplasmic loop, and a short extracellular c-terminus (Albuquerque et al., 2009; Dani, 2015) (Figure 1. A). The second transmembrane domain (M2) of each subunit lines the permeable pore of the receptor, and the M1 and M3 transmembrane domains shield M2 from the cell membrane. The M4 transmembrane domain, on the other hand, has the most contact with the cell membrane (Zoli et al., 2015).

Figure 1. The structure of neuronal nAChRs.

(A) nAChRs are made up of five subunits that come together to form a receptor. Each of these subunits has four transmembrane domains (M1–M4). M2 lines the aqueous pore while M1 and M3 protect M2 from the lipid bilayer. (B) Structure of a homomeric nAChR. These receptors have five agonist binding sites (depicted as pink triangles). (C) Structure of heteromeric α6*nAChRs and agonist binding sites. The X emphasizes that α6*nAChR can potentially combine with other subunits.

nAChR subunits can assemble into either heteromeric receptors containing at least one α and one β subunit (Figure 1. C) or homomeric receptors in which all subunits are identical (Figure 1. B). Homomeric nAChRs have five ligand-binding sites, one between each pair of identical α subunits (Figure 1B). In contrast, most heteromeric nAChRs have two binding sites located between adjacent α and β subunits (Figure 1C), with the exception of α9α10 receptors, which have binding sites between two α subunits, similar to homomeric receptors (Gotti et al., 2006; Zoli et al., 2018). The remaining subunit in heteromeric receptors is an accessory receptor that is not involved in ligand binding but is involved in activation and deactivation of the receptor (Changeux, 1981; Changeux et al., 1984; Gotti et al., 2009). The specific arrangement of subunits within each receptor is highly regulated and dictates its unique pharmacology and function (Wills et al., 2022). The diverse homomeric and heteromeric nAChR subunit compositions contribute to a variety of functions in the central and peripheral nervous systems. In this review, however, we will focus on the understudied α6 subunit.

1.2. Distribution of the α6 subunit

While nAChRs are widely distributed throughout the brain, exhibiting distinct receptor compositions in different regions, α6*nAChRs stand out due to their limited expression compared to some of the most abundant nAChRs. A variety of α6*nAChRs subunit combinations have been reported across many studies. These have shown the α6 subunit mRNA to be predominantly expressed in the locus coeruleus (LC), VTA, and substantia nigra (SN), with additional, lower expression in the reticular thalamic nucleus, supramammillary nucleus, inferior portion of ventral medial habenula (MHb), mesencephalic V nucleus, rostral subnucleus, and caudal subnucleus, as well as retinal ganglion cells, visual thalamus, superior colliculus, and the lateral geniculate nucleus of rodents (Le Novere et al., 1996; Azam et al., 2002; Champtiaux et al., 2002; Gotti et al., 2005; Mackey et al., 2012; Shih et al., 2014; Berry et al., 2015; Barloscio et al., 2017). nAChR composition studies in the dorsal nigrostriatal and ventral mesolimbic dopamine pathways have shown that about 90% of dorsal striatum α6*nAChRs co-express α4 suggesting the presence of both α6β2 and α4β2 binding interfaces in this region (Gotti et al., 2010). In the ventral striatum, about 60% of α6*nAChRs are composed of two α6β2 binding interfaces (Gotti et al., 2010). In addition to regional expression patterns, understanding the combinatorial patterns of the α6*nAChRs within reward-related regions is crucial for investigating the functional and pharmacological differences that influence substance use and other related conditions.

Recent work using fluorescent in situ hybridization has shed new light on the distribution of nAChR subunits in different cell types (dopaminergic, GABAergic, glutamatergic, and noradrenergic) of the VTA and LC of adult and adolescent C57BL/6J mice (Gao et al., 2024). Previous findings demonstrating α6 transcript expression with α4, β2, and β3 transcripts in roughly 90% of VTA dopaminergic neurons were confirmed (Azam et al., 2002 and 2007; Champtiaux et al., 2003). It was further proposed that VTA nAChRs are composed of two primary subtypes: α4α5α6β2β3 and α4α6β2β3, with each accounting for approximately half of the total nAChR population (Gao et al., 2024). Notably, α6 subunit expression was previously believed to be restricted to VTA dopaminergic neurons (Charpantier et al., 1998). However, recent work challenges that assumption by identifying α6 transcripts in roughly half of both GABAergic and glutamatergic neurons within the VTA (Steffenson et al., 2018; Wadsworth et al., 2023; Gao et al., 2024), highlighting a broader distribution than previously recognized. While the α6 subunit had been previously identified in the LC of adolescent rats (Le Novere et al.,1996), the specific cell types expressing it were not determined. New findings now reveal α6 expression in GABAergic, glutamatergic, and noradrenergic neurons within the LC (Gao et al., 2024). Furthermore, noradrenergic neurons may express α6(non-α4)β2β3 nAChRs, whereas GABAergic and glutamatergic neurons potentially express both α4α6β2β3 and α6β2 (Yang et al., 2011; Gao et al., 2024).

While neuronal nAChRs have been extensively researched, their expression in non-neuronal cells remains understudied and is valuable to gain a holistic overview of potential mechanisms underlying drug reward. Growing evidence supports the involvement of astrocytes in substance use disorder, specifically in drug seeking, extinction, and relapse (Scofield, 2018; Linker et al., 2019; Giacometti et al., 2020; Kruyer & Kalivas, 2021; Wang et al., 2022; Tizabi et al., 2024). Mouse astrocytes within the VTA express α4, α6, β2, and β3 transcripts suggesting the potential for several subunit compositions of α6*nAChRs and a potential role for astrocytes in cholinergic system regulation (Gao et al., 2024). Additionally, age- and sex-related differences in nAChR expression were observed with higher relative α6 expression within VTA astrocytes of male adults compared to adolescents. Age and sex differences are critical to evaluate since adolescence is a time of vulnerability to nicotine use and drug addiction and a significant period for the maturation of dopaminergic neurons (Azam et al., 2007; Yuan et al., 2015; Leslie, 2020). This also emphasizes the importance of sex effects in studying α6*nAChRs and how the distribution patterns may differ for each sex. Sex differences have been observed in the neuronal excitability of the MHb, with adult males exhibiting heightened excitability compared to females when exposed to nicotine and menthol (Olszewski et al., 2024). In MHb, α6 has been detected with β2 and β3 suggesting the receptor composition of α6β2β3 (Shih et al., 2014). It has been suggested that combined exposure to high-dose nicotine and menthol may lead to a reduction in α6β2β3*nAChRs within this nucleus (Olszewski et al., 2024). This shift in receptor population may explain the observed increase in neuronal excitability threshold in male subjects and highlight sex-specific variations in nAChR populations within the MHb, consistent with prior studies that have demonstrated sex differences in nAChR populations (Verplaetse et al., 2018; Moen & Lee, 2021).

Taken together, recent work has advanced our understanding of α6*nAChRs expression patterns (Figure 2; Table 1). We now recognize that these receptors are not limited to catecholaminergic neurons but are also expressed in a wider variety of neuronal and non-neuronal cell types, opening new avenues for therapeutic targeting. However, future studies expanding to other brain regions are essential for a comprehensive understanding of these receptor roles, along with more detailed analysis of subunit combinations.

Figure 2. Brain region and cell-type specific expression of α6*nAChRs.

Schematic diagram illustrating the predominant α6*nAChR subunit arrangements found in the dorsal and ventral striatum (different shades of green), ventral tegmental area (purple), and locus coeruleus (blue). Different neuronal and non-neuronal cell populations are shown in different colors; Dopaminergic (Blue), GABAergic (yellow), Glutamatergic (green), Noradrenergic (Red), Astrocytes (purple). The X in the VTA region emphasizes that α6*nAChR can potentially combine with other subunits. Agonist binding sites are depicted as pink triangles.

Table 1.

A summary of distribution of various α6*nAChRs in the brain.

α6*nAChRs Subtype	Brain Region	Organism	Supporting Publication	Neuronal Population
α6β2	Ventral and dorsal striatum	Mouse	Wills et al., 2022 Gao et al., 2019 Yang et al., 2011 Gotti et al., 2010 Azam & McIntosh, 2009	Dopamine GABA Glutamate Norepinephrine
	LC	Rat
α6β4	Striatum LC Hippocampus	Mouse	Azam & McIntosh, 2009	Dopamine Norepinephrine
α6β2β3	Striatum Medial Habenula Superior colliculus Lateral geniculate nucleus	Mouse	Wills et al., 2022 Gao et al., 2019 Shih et al., 2014 Grady et al., 2007 Gotti et al., 2005	Dopamine Norepinephrine
α6β3β4	Interpeduncular nucleus	Mouse	Grady et al., 2009	-
α4α6β2	Striatum VTA	Mouse	Gotti et al., 2010 Azam & McIntosh, 2009	Dopamine GABA
α4α6β2β3	VTA Retinal ganglion cells Striatum LC Superior colliculus Lateral geniculate nucleus	Mouse	Gao et al., 2024 Wills et al., 2022 Gao et al., 2019 Mackey et al., 2012 Gotti et al., 2005	Dopamine GABA Glutamate
α4α5α6β2β3	VTA	Mouse	Gao et al., 2024	Dopamine

2. Conventional and novel strategies in studying α6

While homomeric α7 and heteromeric receptors containing α4 and β2 subunits have been extensively studied (Gotti et al., 2006), other subtypes containing α6 remain under-researched. This is due to a combination of factors, including limited research tools, low expression levels of α6*nAChRs, and a lack of selective agonists for this receptor type (Letchworth and Whiteaker, 2011). The high sequence homology between the α6 and α3 subunits (61% amino acid similarity) further complicates research efforts (Le Novere & Changeux, 1995). Additionally, the absence of a specific antibody for the α6 subunit has hindered investigations into its expression patterns (Cardenas et al., 2020). These challenges have resulted in limited understanding regarding the functional role of α6*nAChRs.

α-Conotoxins (α-Cntx), antagonistic peptides derived from cone snail venom, have been instrumental in investigating the role of the α6 subunit in nAChRs (Azam & McIntosh, 2009; Lebbe et al., 2014). Various α-Cntxs can discriminate between different subtypes of nAChRs. For instance, a mutant of α-CntxPeIA, PeIA[S9H,V10A,E14N], offers high specificity, demonstrating a 290-fold preference for α6/α3β2β3 over α6/α3β4 nAChRs (Hone et al., 2012 (a)), highlighting the utility of these analogs in targeting specific nAChR subtypes. Notably, α-CntxMII and its analogs exhibit selectivity for α6-containing nAChRs over α3β2 nAChRs (McIntosh et al., 2004), leading to insights into the involvement of α6β2* and α4α6β2* receptors in striatal and NAc dopamine release (Kulak et al., 1997; Grady et al., 2007; Exley et al., 2008). Furthermore, α-contoxin PIA (α-CntxPIA) is the first conotoxin to distinguish between α6* and non-α6* nAChRs (Dowel et al., 2003; Pucci et al., 2011). While α-Cntx BulA blocks multiple nAChR subtypes, its mutant form, α-Cntx BulA[T5A;P6O], exhibits a significantly higher affinity for the α6β4 subtype compared to the α6β2 subtype (Azam et al., 2010). α-Cntxs have been used widely across both in vitro and in vivo studies exploring α6*nAChRs. While use of α-Cntxs offers substantial mechanistic insight, interpretation of results from in vivo studies must consider their regional administration, limited stability, and issues with bioavailability and crossing of the blood-brain barrier (Armishaw, 2010). The following sections explore other methods of investigating α6*nAChRs and discuss developing novel strategies.

2.1. In Vitro Expression of α6*nAChRs

The potential of drugs targeting α6*nAChRs as therapeutic targets highlights the importance of pharmacological studies in this area. While in vitro systems offer the advantages of rapid, high-throughput experimentation and a smoother transition to in vivo studies, the challenge lies in achieving functional receptor formation of α6*nAChRs. Nevertheless, the discovery of chaperone proteins has opened new avenues for in vitro investigations, providing valuable insights into this promising therapeutic target. Assembly of nAChRs is a highly regulated process and relies on the correct folding of the subunits. The assembled receptors need to be transported from their site of production, the endoplasmic reticulum, to the cell surface (Millar et al., 2008). Some of the challenges in expressing α6*nAChRs in mammalian cells include aggregation of subunits, low receptor binding levels, poor cell surface transport and therefore low cell surface expression (Kuryatov et al., 2000; Evans et al., 2003). As part of efforts to investigate the functional expression of these receptors, it has been established that the Novel Acetylcholine receptor Chaperone (NACHO), a transmembrane protein in the endoplasmic reticulum of neurons, is crucial for the assembly of α6β2β3 receptors, as evidenced by reduced binding sites in NACHO knockout mice (Gu et al., 2016). However, these receptors remain intracellular even with NACHO, suggesting that additional factors are required for their functional expression (Gu et al., 2016; Matta et al., 2017). Subsequently, four accessory proteins necessary for α6β2β3 nAChR expression were identified. In addition to NACHO, β-anchoring and regulatory protein (BARP), lysosomal-associated membrane protein 5 (LAMP5), and a cytosolic sulfotransferase (SULT2B1) are also involved. LAMP5 and SULT2B1 facilitate receptor expression on the cell surface, while BARP is responsible for channel gating (Gu et al., 2019). Furthermore, there are differences in the accessory proteins necessary for the assembly of different α6*nAChRs compositions. Specifically, α6β4 does not necessitate NACHO but instead requires BARP, SULT2B1, and inositol-requiring enzyme-1α (IRE1α), which functions as an unfolded protein response (UPR) sensor and facilitates the assembly of α6β4 receptors through the UPR pathway (Knowland et al., 2023). The function of BARP in α6β4 is different from that of α6β2β3 receptors. BARP regulates the surface transport of these receptors suggesting distinct mechanisms for the regulatory role of BARP in different α6*nAChRs (Knowland et al., 2023). With the path now cleared for high-throughput screening, these discoveries will translate into a deeper understanding of α6*nAChRs in vitro, serving as a crucial step toward the development of targeted therapies.

2.2. Animal Models

Animal models have been pivotal in advancing our understanding of α6*nAChRs and helped establish the fundamental findings of these receptors’ function. Early work using knockout models have demonstrated the role of α6*nAChRs in nicotine-induced dopamine release and self-administration (Champtiaxu et al., 2003; Pons et al., 2008). The α6-GFP mouse model, where there is a GFP fused in-frame in the M3–M4 intracellular loop of the subunit, has been a valuable tool that allowed for high-resolution visualization of α6*nAChRs in the mouse visual system (Mackey et al., 2012). Given the broader expression of α6*nAChRs, including in astrocytes (Gao et al., 2024), this α6-GFP mouse model holds immense potential for mapping their expression patterns. Another useful animal model in the history of studying α6*nAChRs is the gain-of-function α6^L9’S in which leucine 9 of M2 transmembrane (leucine 280) has been mutated to serine by homologous recombination (Drenan et al., 2008). This mutation has been shown to increase nicotine-induced locomotion activity even at a low dose (0.15 mg/kg, i.p.), illustrating a high-sensitivity function for α6^L9’S nAChRs (Drenan et al., 2008). This model is particularly promising for investigating locomotor activity and dopamine release in response to novel α6*nAChRs agonists and antagonists. Such innovative approaches have also been applied to other nAChR subunits (Lester et al., 2003; Drenan & Lester, 2012; Lotfipour et al., 2017; Walker et al., 2023; Wells et al., 2024).

Genomic studies have linked single nucleotide polymorphisms (SNPs) within the CHRNA6 gene to conditions such as drug addiction. One such SNP, rs2304297, located in the 3’ untranslated (UTR) of the CHRNA6 gene, has been associated with enhanced smoking behavior in humans (Table 2). To investigate the role of this SNP, a novel transgenic animal model was developed by replacing the rat CHRNA6 3’ UTR with the human version, creating two “humanized” rat lines: one homozygous GG (α6^GG) and one homozygous CC (α6^CC) (Cardenas et al., 2022). The clinical and pre-clinical findings for this SNP will be further discussed in subsequent sections. Collectively, these animal models exemplify how invaluable they are for investigating the functional impact of SNPs. The models discussed here represent just a few examples of the powerful tools available for continued research or inspiration for the creation of novel models to further elucidate the role of α6*nAChRs.

Table 2.

Clinical studies of the rs2304297 SNP, located in the 3’ UTR of the CHRNA6, associated with nicotine/tobacco use.

Supporting Publication	Adolescent	Sample Size	% Male	“G” as Risk Allele
Pugach et al., 2017	Yes	480	40	Yes
Fontana et al., 2015	No	12507	40.9	No%
Cannon et al. 2014	Yes	439	41	Yes
Fletcher et al., 2012	No	6178	48	No%
Pedneault et al., 2014	Yes	1293	35	No#
Lee et al., 2012	No	789	48.9	Yes*
Thorgeirsson et al., 2010	No	76681	Not Reported	Yes*
Lotfipour et al., 2010	Yes	423	45.4	Yes
Hoft et al., 2009	Yes	1051	49.2	Yes
Hoft et al., 2009	Yes	1051	49	No+
Dawes et al., 2009	Yes	2674	48	Yes
Zeiger et al., 2008	Yes	1056	58.1	Yes
Saccone et al., 2007	No	1929	38	Yes
Rigbi et al., 2008	No	244	0	Yes^

^*Does not survive multiple comparison correction;

^#Carriers of minor allele (C) report decreased dizziness at first inhalation;

^%GG protective in the presence of higher taxes;

⁺C not G listed as risk allele;

^{^}G is a risk genotype.

2.3. Computational Methods

The utilization of lead compounds and computational modeling represents a well-established approach in drug discovery, and it has undergone significant refinement and advancement in recent years. This strategy leverages existing knowledge of compounds with known activity against a target, coupled with the computational power of protein modeling, virtual screening, and molecular dynamics simulations, to expedite the identification of promising drug candidates. A prime example of this approach lies in targeting the α6β2*nAChR receptor, implicated in nicotine addiction. Given the side effects of current treatments such as depression, psychosis, and suicide (Prochaska & Benowitz, 2019), this receptor subtype represents a promising avenue for novel therapies. Researchers have employed computational methods, using Indolizidine (−)-237D, a known selective inhibitor, as a lead compound to screen over 2000 drugs. This has led to the identification of eight potential antagonists which need further investigation (Acquah et al., 2021). Although in vitro and in vivo validation remains essential, the integration of lead compounds and computational modeling offers a powerful strategy to significantly reduce the time and cost associated with initial drug discovery investigations.

2.4. Omics Approaches

The study of subunit distribution, particularly for those with low expression levels like α6, has been hindered at the protein level by technical limitations such as the lack of specific antibodies (Letchworth & Whiteaker, 2011; Cardenas et al., 2019). However, advancements in transcriptomics and proteomics offer new avenues for investigation in addiction neuroscience and nAChR biology. Methods like RNA sequencing, polysome profiling, and ribosome profiling allow researchers to probe transcripts directly, providing insights into gene expression, translation, and regulation that complement traditional protein-based approaches. These approaches have already helped reveal mechanisms underlying age and sex differences in substance use. Coupling a low dose of nicotine exposure paradigm that models smoking “initiation” with a NanoString multiplex gene panel, an mRNA-based approach, showed that nicotine increased cocaine self-administration, decreased NAc inflammatory CX3CL1 transcripts, and increased NAc dopamine D2 receptor transcripts in adolescent rats (Linker et al., 2020). Further evaluation revealed that NAc D2 receptors and CX3CL1 are a mechanistic interface for nicotine-induced microglial activation with resulting pruning of glutamate synapses and enhanced cocaine reinforcement in adolescents (Linker et al., 2020). Large scale patterns of protein expression using mass spectrometry in mice have also revealed baseline, nicotine-, and cocaine-induced sex differences in the NAc and VTA despite similar behavioral performance (Lee et al., 2021; López et al., 2021). Further investigation into unique pathways identified in studies such as these and focusing efforts on specific nAChR subunits can identify novel therapeutic targets. Given potential difficulties in identifying nAChR subunits at the protein level, ribosome profiling may serve as a powerful tool as it offers a global snapshot of translation (Ingolia et al., 2019), enabling the identification of actively translated subunits such as α6. By integrating these newer techniques combined with established methods, we can gain a comprehensive understanding of potential receptor subunit compositions across diverse brain regions and cell types.

3. α6*nAChRs: Function and Impact

The diverse combinations of nAChR subunits are the primary determinants of both their function and pharmacological properties, including ion selectivity, signal intensity, desensitization, and recovery speed (Dani, 2015). These receptors play crucial roles in both excitatory and inhibitory neurotransmission (Albuquerque et al., 2009), impacting cognitive function, synaptic plasticity, learning and memory, and reward pathways (Jones et al., 1999; Hurst et al., 2013; Galvin et al., 2020; Koukouli & Changeux, 2020; Mineur et al., 2023). α6*nAChRs, particularly within the dopaminergic neurons of the mesolimbic pathway, have a major role in modulating dopamine release in the striatum (Drenan et al., 2010). Dopamine activity within the mesolimbic pathway is highly implicated in drug response and the development of drug dependence (Volkow & Morales, 2015). Indeed, there is a wealth of literature illustrating that α6*nAChRs are crucial in mediating the addictive properties of stimulants such as nicotine and alcohol (Drenan et al., 2008; Kuzmin et al., 2009; De Biasi & Dani, 2011; Gao et al., 2019). These receptors are associated with nicotine-induced dopamine release and locomotor activity in rats (Le Novere et al., 1999; Drenan et al., 2008; Zoli et al., 2015), as well as nicotine self-administration (Pons et al., 2008; Brunzell et al., 2010; Gotti et al., 2010). In vitro studies using mouse primary dopamine neuron cultures and iPSC-derived human dopamine neurons have shown that α6*nAChRs mediate nicotine-induced structural plasticity (Collo et al., 2018). Neuroplasticity and synapse formation are other critical functions involved in substance use and dependence (Robinson & Kolb, 2004; Russo et al., 2010; Natividad et al., 2019; Linker et al. 2020), with important implications for age- and sex-related differences in maturation of the dopamine system and drug response (Azam et al., 2007). While these studies offer valuable insight into the complex functions of α6*nAChRs, many questions remain unanswered. This section will delve deeper into the drug-related functions of these receptors, paving the way for a more comprehensive understanding of their therapeutic potential.

3.1. Nicotine

3.1A. α6*nAChRs and Dopamine Release

Nicotine exerts its rewarding effects by acting on nAChRs within the mesolimbic dopamine pathway (Ebrahimi et al., 2024). Specifically, nAChRs on dopamine neurons in the VTA, which project to the nucleus accumbens (NAc), trigger dopamine release, a key factor in the addictive properties of psychostimulants like nicotine (De Biasi & Dani, 2011) (Figure 3). Given the diverse subunit composition of nAChRs in the mesolimbic dopamine pathway, identifying the specific subtypes involved in dopamine release has been a central research question. Notably, α6*nAChRs, predominantly localized to dopaminergic neurons (Azam et al., 2002; Azam & McIntosh, 2006), have been shown to modulate nicotine-induced dopamine release, particularly in the ventral striatal NAc compared to the dorsal striatal caudate putamen (Exley et al., 2008). Additionally, intra-VTA perfusion of α-CntxMII, selective for α3/α6β2, and α-CntxPIA, selective for α6β2, suppress nicotine-induced dopamine release in the NAc (Gotti et al., 2010; Zhao-Shea et al., 2011). The VTA region can be divided into three subregions: anterior VTA (aVTA), posterior VTA (pVTA), and tail VTA (tVTA) (Zhao-Shea et al., 2011). Investigating the expression pattern of nAChR subunits in these regions has led to the finding that pVTA has significantly higher expression of α4, α6, and β3 subunits in comparison to aVTA dopaminergic neurons (Zhao-Shea et al., 2011). The crucial role of pVTA in mediating the rewarding properties of nicotine and its dopamine release was further illustrated when injection of a noncompetitive nAChR antagonist, mecamylamine, blocked these nicotine effects (Zhao-Shea et al., 2011). Further studies in knock-out and wild-type mice demonstrated the importance of both α4 and α6 subunits in the modulation of dopaminergic neurons and suggests that these effects are mediated by α6α4β2*nAChRs in the pVTA (Gotti et al., 2010; Zhao-Shea et al., 2011; Liu et al., 2012).

Figure 3. Nicotine’s effects on the mesolimbic dopamine system.

Nicotine binds to the α6*nAChRs on dopaminergic neurons in the posterior ventral tegmental area (pVTA), leading to dopamine release in the nucleus accumbens (NAc). This dopamine release reinforces the rewarding properties of nicotine and increases locomotor activity, contributing to nicotine dependence. Injection of mecamylamine, an nAChR antagonist, and α-Ctx-PIA, an α6*nAChR antagonist, into the pVTA blocks dopamine release and therefore the reinforcing properties of nicotine and locomotor activity, respectively.

3.1B. α6*nAChRs and Locomotor Activity

One of the behavioral outputs of dopamine release in rodents is locomotor activity (Picciotto et al., 1998). Oligonucleotides against the α6 subunit result in a partial inhibition of nicotine-induced locomotion, suggesting the involvement of α6*nAChRs (Le Novere et al., 1999). The highly specific α-CntxPIA infused into the pVTA inhibits nicotine-induced locomotion and dopamine release (Gotti et al., 2010). These effects suggest that α6*nAChRs are necessary and sufficient for nicotine-induced locomotor activity. Indeed, this is corroborated by studies revealing enhanced nicotine-induced locomotor activity via α6*nAChRs (Drenan et al., 2008; Moen et al., 2021). The hypersensitive α6^L9’S mouse line was used to demonstrate increased baseline locomotion compared to wild-type mice (Drenan et al., 2008). Additionally, a dose-dependent increase in locomotor activity in response to nicotine (0.02–0.4 mg/kg, IP) was seen, which was blocked by the injection of α-CntxMII into the VTA (Berry et al., 2015). Administration of the α6β2 antagonist, N, N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), in male and female mice has induced a decrease in both locomotor activity in females between 10 to 30 minutes post injection (Kamens et al., 2017). Using protein kinase C epsilon (PKCε) knock-out female mice, enhanced nicotine-induced locomotor activity was associated with increased α6 and β3 expression (Moen et al., 2021). PKCε is a critical signaling molecule that regulates the CHRNA6-CHRNB3 cluster (Lee et al., 2015) and may serve as a potential target for pharmacotherapies given its role in modulating nicotine sensitivity. However, it is important to note that the locomotor response to nicotine is not uniform across all mouse strains and experimental conditions. Of note, the previously described findings reflect the effects of low-dose nicotine. Acute exposure of C57BL/6J mice to a high dose of nicotine (1.5 mg/kg) did not alter locomotor activity (Jackson et al., 2009). These discrepancies highlight the complexity of nicotine’s effects and underscore the importance of considering factors such as mouse strain, nicotine dose, and administration route when interpreting experimental results. While the precise mechanisms underlying nicotine’s effects on locomotor activity and dopamine release remain to be fully elucidated, the available evidence points to a crucial role for α6*nAChRs on VTA dopaminergic neurons.

3.1C. α6*nAChRs and Self-Administration

Nicotine self-administration is a critical behavioral measure to consider when investigating neurobiological mechanisms and developing potential treatments for addiction. In a self-administration study using wild-type and α6-knockout (α6-KO) mice, wild-type mice self-administered nicotine (26.3 μg/kg/Inj), whereas α6-KO mice did not, even at higher doses. Notably, re-expression of the α6 subunit in the VTA of KO mice restored nicotine self-administration (Pons et al., 2008). Further investigation has shown that α6β2 receptors on dopamine terminals in the NAc shell are not only necessary for nicotine self-administration, but also for maintaining this behavior, as blockade of these receptors reduced rats’ motivation for reinforcement (Brunzell et al., 2010). Moreover, pretreatment with α-CntxMII in the VTA, blocking α6*nAChRs, decreased nicotine self-administration responding in rats (Gotti et al., 2010). Systemic administration of the α6β2 antagonist, bPiDI, also impairs acquisition and maintenance of nicotine self-administration (Beckmann et al., 2015; Maggio et al., 2018). Collectively, these findings highlight the α6 subunit as a critical regulator of the reinforcing effects of nicotine and nicotine intake and may be a reliable target for smoking cessation therapies.

3.1D. Clinical and Pre-Clinical Studies Investigating CHRNA6, Encoding for α6 Subunit

Clinical research has established a strong genetic component to nicotine dependence, with heritability estimates ranging from 40–70% (Li et al., 2003; Maes et al., 2004; Sharp & Chen, 2019). As nicotine is an agonist for nAChRs, genes encoding these receptors are prime candidates for investigation. In particular, identifying SNPs within nAChR genes and their association with smoking behavior is crucial for understanding the genetic underpinnings of nicotine dependence. While a SNP in the coding region of the CHRNA5 gene, encoding the α5 nAChR subunit, has been previously studied and linked to nicotine dependence (Bierut et al., 2008; Morel et al., 2014), other nAChR subunits, such as α6, warrant further scrutiny. One study examined the SNPs in the CHRNB3-CHRNA6 gene cluster in a Chinese Han population. Various forms of analysis, such as haplotype analysis, gene-gene interactions, and meta-analysis, were employed to investigate the genetic factors underlying nicotine addiction. These analyses confirm that both genes within this cluster contribute significantly to nicotine dependence (Wen et al., 2017). For CHRNA6, rs2304297, a SNP in the 3’ UTR of the gene has been found to be associated with nicotine dependence in Caucasians (Saccone et al., 2007; Hoft et al., 2009a). There is an abundance of evidence associating this SNP with nicotine-seeking behaviors and susceptibility to nicotine addiction in humans, particularly adolescents (Table 2) (Lotfipour et al., 2010; Thorgeirsson et al., 2010). While most studies listed in Table 2 identify the “G” allele as the risk allele, some report it as protective. This SNP has also been associated with increased positive perception of nicotine during adolescence and an increase in daily cigarette use (Table 2) (Zeiger et al., 2007; Pedneault et al., 2014).

Given the substantial clinical evidence linking this SNP (rs2304297) to nicotine dependence, our laboratory has created two rat lines facilitating pre-clinical investigations into the SNP’s functional role and elucidating its contribution to nicotine addiction. In the rat lines, the 3’ UTR of the CHRNA6 gene of rat was replaced with that of humans. Using sub-chronic, low-dose nicotine exposure as a model of early adolescent nicotine use, sex and genotype differences were observed, with female α6^CC and male α6^GG exhibiting increased locomotor and anxiolytic behavior compared to saline controls (Cardenas et al., 2022). Furthermore, adolescent male α6^GG were more prone than male α6^CC to relapse into nicotine-seeking behavior after drug plus cue exposure (Carreño & Lotfipour, 2023). In α6^CC females, sub-chronic nicotine treatment also resulted in the ability to discriminate between reinforced and non-reinforced responses in a methamphetamine self-administration paradigm (Carreño et al., 2024) (Table 3).

Table 3.

Summary of pre-clinical findings of the rs2304297 SNP

Behavioral Measure	Male Rats α6GG or α6CC	Female Rats α6GG or α6CC
Locomotor Activity and Anxiolytic Behavior	Increased locomotor activity and anxiolytic behavior in nicotine-treated male α6GG compared to saline controls Increased anxiolytic behavior in nicotine-treated male α6GG compared to male α6CC	Increased locomotor activity and anxiolytic behavior in nicotine-treated α6CC compared to saline controls as well as nicotine-treated α6GG
Dopamine Release	Increased dopamine overflow in response to nicotine and methamphetamine in α6GG compared to α6CC	Increased dopamine overflow in response to nicotine and methamphetamine in α6CC compared to α6GG
Methamphetamine Self-administration	No genotype effect observed in meth self-administration after exposure to nicotine	Enhanced discrimination between reinforced and non-reinforced responding in nicotine-treated α6CC and saline-treated α6GG
Tissue Neurotransmitter Levels	Nicotine + cue treated adolescent α6GG rats had decreased dopamine levels in the NAc shell compared to other groups Baseline differences: drug-naïve adolescent α6GG rats had greater dopamine levels in the NAc core compared to their adult counterparts, and adolescent α6CC	No differences were observed

In preliminary neurochemical studies, exposure to nicotine and methamphetamine increased dopamine overflow in α6^GG males and α6^CC females compared to their genotype counterparts (Carreño et al., 2024) (Table 3). Tissue neurotransmitter levels were also measured in drug-naïve adults and adolescents, carrying the CHRNA6 3’ UTR SNP revealing baseline age and genotype differences in DA levels (Carreño et al., 2024). Adolescent male α6^GG rats had significantly greater DA levels in the NAc core than their adult counterparts, and adolescent male α6^CC rats. No genotype or age differences were observed in females. The combined behavioral and neurochemical work using this model provides important insight on novel mechanisms in nicotine-induced behaviors driven by a SNP in an UTR of a nAChR subunit gene. While the precise mechanisms remain to be elucidated, these findings reveal new insights into how this SNP drives nicotine-induced behaviors.

3.2. Alcohol

The frequent co-use of alcohol and tobacco in humans is well established (Daeppen et al., 2000; John et al., 2003; Grant et al., 2004). Indeed, individuals with alcohol use disorder (AUD) often have nicotine dependence (Weinberger et al., 2020; Frie et al., 2022). Bidirectional associations between nicotine and alcohol have been described across several studies (Cannizzaro et al., 2022; Patrick et al., 2023). Although the field has put forth effort in evaluating these drugs, alone or combined, there is still limited understanding of underlying mechanisms or therapeutic treatments (Morikawa & Morrisett, 2010). Human genetic variations in CHRNA6, encoding for α6 subunit, have been linked to smoking and alcohol in some instances (Table 2), and supports further investigation of the role of these receptor subunits in alcohol use and vulnerability.

Unlike nicotine, alcohol is not a direct agonist for nAChRs, thus suggesting an alternative modulatory role influencing how nAChRs respond to stimuli and regulate neurotransmitter release (Rahman et al., 2015). Injection of mecamylamine, a non-selective nAChR antagonist, into mouse VTA attenuates ethanol-induced increases in dopamine levels in the NAc (Blomqvist et al., 1997; Larsson et al., 2002 & 2004). nAChRs are also associated with alcohol-related behaviors such as sedation (Kamens et al., 2012), locomotor activity (Larsson et al., 2002; Kamens et al., 2017), and intake (Feduccia et al., 2012; Kamens et al., 2018). Specifically, α6*nAChRs are necessary for ethanol-induced activation of VTA dopamine neurons and increased ACh release in mouse tissue slices (Larsson et al., 2002; Liu et al., 2013). Low dose (0.1–5 mM) ethanol-induced dopamine release in the NAc can be blocked using α-CntxMII, suggesting ethanol acts as an allosteric modulator on α6*nAChRs (Gao et al., 2019). Transgenic mice with heightened nicotine sensitivity, α6L9′S, also show enhanced ethanol sensitivity, alcohol intake and conditioned place preference (Powers et al., 2013), suggesting a role for the ɑ6 subunit in mediating ethanol’s neurochemical and behavioral effects. Indeed, other animal models also indicate that α6*nAChRs regulate alcohol-induced dopamine release, locomotor activity, and voluntary intake in mice (Powers et al., 2013; Wang et al., 2014; Gao et al., 2019). Collectively, these studies strongly suggest that α6*nAChRs modulate alcohol-induced dopamine function within the mesolimbic pathway with subsequent impacts on alcohol-related behaviors.

While there is clear overlap in α6 subunit involvement for both nicotine- and alcohol- related dopamine function, a growing body of literature strongly implicates regional and circuit level influence of GABA (Steffensen et al., 2018; Gao et al., 2019; Wadsworth et al., 2023). α6*nAChRs are expressed on GABAergic terminals projecting onto both dopamine and GABAergic neurons within the VTA (Yang et al., 2009; Steffensen et al., 2018; Gao et al., 2024). α6*nAChRs on the GABAergic terminals to VTA GABA neurons enhance low-dose ethanol-induced dopamine release in the NAc and subsequent ethanol conditioned place preference (Schilaty et al., 2014; Steffenson et al., 2019) but inhibit these effects at high ethanol doses (Gao et al., 2019). Taken together, the findings suggest that ethanol, at low doses, enhances GABA transmission onto VTA GABA neurons and increases overall activation of VTA dopamine neurons with parallel downstream increases in dopamine release within the NAc (Steffensen et al., 2018; Gao et al., 2019; Wadsworth et al., 2023).

While α6*nAChRs are strongly implicated in mediating ethanol’s rewarding effects, the precise mechanisms by which high dose ethanol inhibits evoked dopamine release while low doses enhance spontaneous dopamine release remains unclear. Discrepancies in species, ethanol doses, neuronal population, and experimental paradigms across studies complicate the interpretation and integration of findings. Moreover, the focus on dopaminergic neurons and the use of electrophysiological techniques in many studies limits our understanding of the complex interplay between different neurotransmitter systems in response to ethanol. Comprehensive investigations examining the interactions between dopaminergic and GABAergic neurons within different brain circuits are necessary to elucidate the full picture of ethanol’s effects on the brain.

3.3. α6 subunit in Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the nigrostriatal pathway, containing both α4β2* and α6β2*nAChR subtypes (Grady et al., 2007; Gotti et al., 2009; Halliday et al., 2011; Rascol et al., 2011). A major function of the nigrostriatal dopaminergic system is to facilitate motor activity. In PD, damage to nigrostriatal neurons results in reduced α4β2*- and α6β2*nAChR-mediated dopamine release, leading to the characteristic symptom of loss of motor function. (Pérez et al., 2010; Pérez & Quik, 2011). A prevalent therapeutic intervention employed for Parkinson’s disease patients is levo-dihydroxyphenylalanine (L-DOPA), which serves as a precursor for dopamine. The co-administration of L-DOPA along with carbidopa has demonstrated efficacy in alleviating the motor symptoms (Hansen et al., 2022) (Figure 4). L-DOPA crosses the blood-brain barrier and is converted to dopamine whereas carbidopa inhibits DOPA decarboxylase and prevents its metabolism (Hansen et al., 2022). This treatment can lead to L-DOPA-induced dyskinesia (LID) within months of starting therapy (Manson et al., 2012; LeWitt, 2015). The elucidation of the molecular mechanisms underpinning LID holds significant value and could potentially facilitate the optimization of L-DOPA therapy. Whereas nicotine inhibits LID via nAChR desensitization, this effect is eliminated in mice with the α6 subunit deleted (Quik et al., 2012; Bordia et al., 2015). A study with α6L9’S hypersensitive mice, further emphasizes the critical role of α6*nAChRs in LID, suggesting a possible therapeutic role for α6β2*nAChR-selective drugs in treating LID (Quik et al., 2011; Bordia et al., 2015).

Figure 4. Illustration of L-DOPA therapy and potential role of AN6001 in Parkinson’s patients.

(A) The nigrostriatal pathway, crucial for motor control, with dopamine neurons projecting from the substantia nigra (SN) to the striatum. Degeneration of these neurons (shown in blue) in Parkinson’s causes dopamine deficiency and motor dysfunction. (B) L-DOPA, a precursor to dopamine, crosses the blood-brain barrier (BBB) and is converted into dopamine, offering symptomatic relief. However, L-DOPA therapy can lead to L-DOPA-induced dyskinesia (LID), which is linked to α6β2*nAChRs (shown in purple). (C) The positive allosteric modulator, AN6001(red triangle), selectively enhances α6β2*nAChR activity, offering a potential avenue for neuroprotection and alleviating LID. AN6001’s targeted action on α6β2*nAChRs may provide a more refined therapeutic approach compared to non-specific agonists.

Various studies have suggested that nicotine, despite its well-known risks, may offer unexpected neuroprotective benefits, particularly in the context of PD. Both in vitro studies on dopaminergic neurons and in vivo studies on PD animal models indicate that nicotine can protect against cell death and nigrostriatal damage, respectively (Quik et al., 2007; Bordia et al., 2008; Picciotto & Zoli, 2008; Zhang et al., 2013). In the 6-hydroxydopamine-induced nigrostriatal lesion model of PD, both α4β2*- and α6β2*-mediated dopamine release is progressively lost with increased nigrostriatal damage (Perez & Quik, 2011). Although nicotine is neuroprotective it is non-specific, interacting with all types of nAChRs. A novel α6β2*nAChR positive allosteric modulator, AN6001, has been shown to protect dopaminergic neurons from neurotoxic damage in vitro, and has been suggested as a potential therapy for treatment of PD (Van Hout et al., 2020) (Figure 4). Positive allosteric modulators have reduced chances of side effects as they do not directly activate or overstimulate receptors. Additionally, they exhibit greater selectivity for specific receptor subtypes due to the less conserved nature of allosteric sites compared to the main binding site (Wang et al., 2018). In this case, AN6001 exhibits significantly higher selectivity for α6β2* compared to α4β2* nAChRs, one of the most abundant nAChRs in the brain (Hout et al., 2020). Although AN6001 has demonstrated potential in HEK293 cells and Xenopus oocytes, its efficacy must be evaluated in PD animal models to advance its therapeutic development.

3.4. α6 subunit in Pain

Chronic pain patients often experience spontaneous pain and heightened sensitivity to normally non-painful stimuli, such as light touch (mechanical allodynia) (Fillingim et al., 2016; Inoue, 2021; Dong et al., 2022). P2X2/3 receptors are ionotropic receptors that are involved in the development of neuropathic pain (Dong et al., 2022). Current treatments for this condition are ineffective, and we lack a thorough understanding of the underlying molecular mechanisms of symptoms such as mechanical allodynia. nAChRs have been identified and investigated as a potential drug target for chronic pain and attention has been on α4β2*, α7*, α9*, and α3* nAChRs (Papke & Horenstein, 2021; Zheng et al., 2021; Hone & McIntosh, 2023). Dorsal root ganglia (DRG), which contain neurons responsible for transmitting sensory information to the central nervous system, have been found to express α6β4 nAChRs in rats (Hone et al., 2012 (b)). Immunohistochemistry of mouse DRG α6-containing neurons have also shown nociceptive neuron markers (Wieskopf et al., 2015). To better understand the molecular mechanisms, the genetic basis of mechanical allodynia has been examined using genome-wide gene expression levels in DRG of 25 inbred mouse strains (Wieskopf et al., 2015). The results suggest the involvement of the CHRNA6 gene and α6*nAChRs in influencing mechanical allodynia following nerve damage or inflammation. The proposed analgesic mechanism of action of α6* receptors is interaction with, and inhibition of, P2X2/3 receptors (Wieskopf et al., 2015). Additionally, the antiallodynic effect of nicotine has been investigated using α4 and α6 KO as well as wild type mice (Wieskopf et al., 2015). Nicotine was administered via three types of administration, supraspinal, spinal, and peripheral in both neuropathic and inflammatory pain models. The study illustrated that nicotine’s ability to alleviate mechanical allodynia is primarily mediated through α6*nAChRs, with the exception of supraspinal regions where both α6 and α4 receptors seem to play a role (Wieskopf et al., 2015). The α6 subunit, once overlooked, emerges from this study as a key player in pain processing, highlighting its potential as a therapeutic target.

Successful heterologous expression of α6β4*nAChRs has paved the way for further investigation and examination of the involvement of this receptor subtype in pain. The efficacy of two agonists, ABT-594 and ABT-894, has been tested on α6β4 and α4β2 receptors expressed in HEK293T cells. Whereas ABT-594 worked equally well on both, ABT-894 was less efficient on α6β4 (Knowland et al., 2020). In vivo testing of ABT-594 in BARP-KO mice which have reduced expression of α6β4 has shown blunted antiallodynic effects as compared to wild-type controls (Knowland et al., 2020). Although in vitro and in vivo studies have illuminated the role of α6β4*nAChRs in pain, bridging the gap to human applications remains crucial. Species-specific differences, such as those observed in α-conopeptide binding of human and rat α6β4*nAChRs (Hone et al., 2021), highlight the complexities of translating preclinical findings to clinical applications.

Genetic variations within the CHRNA6 gene have been studied in pain clinical studies (Wieskopf et al., 2015). More specifically, a rs7828365 SNP within the promoter region was shown to be linked to altered pain sensitivity, such that homozygous individuals with the minor allele (TT) were more likely to experience persistent pain six months after surgery than subjects with TC and CC. Whereas this suggests a role for this gene and α6*nAChRs in pain in both humans and mice, the authors acknowledge the infrequent occurrence of this genotype among humans and that other factors besides CHRNA6 likely contribute to the clinical variability of chronic pains. Additionally, the small number of homozygous TT individuals posed significant challenges and reduced the statistical power of the study. Therefore, a larger cohort of individuals would be beneficial in further understanding the role of CHRNA6 in chronic pain (Wieskopf et al., 2015). Collectively, the findings suggest that α6β4-containing receptors play a role in pain modulation. However, future research is necessary to fully elucidate the precise mechanisms involved and to identify additional compounds that could effectively target this receptor for pain relief.

Conclusion

Nicotine and other substance use disorders impose several threats to public health and exert a tremendous individual and socioeconomic strain. There is an urgent need to uncover neurobiological mechanisms mediating substance use and gather a comprehensive understanding of the role of nAChRs as therapeutic targets. In this review, we outline the key role of α6*nAChRs in nicotine and alcohol use and the genetic factors which may influence their function. We also review implications for α6*nAChRs in alleviating dopamine-related side effects in PD treatment and potential considerations for α6*nAChRs in pain conditions. Historically, challenges like low expression levels and limited research tools have hindered the investigation of α6*nAChRs compared to other nAChR subtypes (Letchworth & Whiteaker, 2011). However, this review highlights recent advancements in research tools which have enabled significant progress. Improved methods for in vitro expression of functional and physiologically relevant α6*nAChRs will provide valuable insight into basic mechanisms of action for substances and development of effective treatments. High-throughput screening, using either in vitro or in vivo models, is a promising area of exploration for addiction research as it accelerates drug discovery and saves valuable time before pre-clinical and clinical trials. Furthermore, studies using innovative animal models underscore their indispensable role in understanding these receptors and potential cessation strategies.

Once believed to be restricted to dopaminergic neurons, α6*nAChRs are now known to be expressed in GABAergic, glutamatergic, and noradrenergic neurons (Steffenson et al., 2018; Wadsworth et al., 2023; Gao et al., 2024). This challenges previous research that focused solely on dopaminergic neurons and suggests a broader role for α6*nAChRs in cellular functions at the circuit level and beyond. Here, we review work focused on the impact of α6*nAChRs on VTA GABA neurons under various ethanol conditions. Future studies could evaluate the role of α6*nAChRs in the context of other substances and combined exposure such as nicotine and alcohol. The discovery of α6*nAChR expression in astrocytes, non-neuronal cells implicated in drug addiction, opens exciting avenues for research, particularly given the growing evidence linking these receptors to substance use disorders (Morikawa & Morrisett, 2010; De Biasi & Dani, 2011; Ebrahimi et al., 2024).

The adolescent brain is especially susceptible to the effects of nicotine (Leslie, 2020), and it is essential to investigate how these receptors differ between sexes as well. Nicotine-induced dopamine levels in the NAc are greater in adolescent rats than in adult rats (Corongiu et al., 2020), and adolescent females show increased sensitivity to its rewarding effects (Xue et al., 2018). α6 subunit mRNA expression peaks in VTA and substantia nigra dopamine neurons during adolescence in male rats (Azam et al., 2007), thus it is critical to evaluate how age impacts α6*nAChR function and substance use. Sex differences in α6 subunit mRNA expression have been observed in VTA astrocytes and MHb neurons (Gao et al., 2024; Olszewski et al., 2024). As reviewed in Meon et al. (2021), there are clear sex differences in nAChR expression patterns throughout the brain in adults with various implication in both nicotine and alcohol related behaviors. However, it is unclear exactly how α6*nAChR differs between sexes throughout development and the extent of their role in behavior. Thus, sex and age differences are critical considerations to be implemented into future studies to further explore α6*nAChR distribution and function.

Mounting evidence clearly suggests α6*nAChRs play a key role in reward-related behavior and substance use disorders, particularly nicotine and alcohol use. Polymorphisms in CHRNA6, the α6 subunit gene, are associated with both nicotine and alcohol susceptibility (Schlaepfer et al., 2008; Hoft et al., 2009; Wang et al., 2009; Thorgeirsson et al., 2010). Common genetic factors underlying nicotine and alcohol codependency are important to evaluate since it may influence how a person responds to substances and any cessation treatments. In addition, identifying individuals who are more susceptible to the rewarding effects of abused drugs such as nicotine is critical for prevention strategies. Of note, the smoking cessation treatment with success in treating alcohol dependence, Varenicline, is a potent partial agonist of α4β2-containing and α6β2-containing nAChRs (Bordia et al., 2012). Genetic factors, including the SNPs highlighted in this review, may contribute to individual differences in response to treatments, offering insights into developing more effective treatments. Overall, the accumulating evidence highlights α6*nAChRs as promising therapeutic targets, underscoring the need for further investigation to fully realize their potential in treating various diseases.