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 the 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.

1. Introduction

Nicotine use remains one of the most preventable threats to public health and leading causes of premature death in the United States [1]. 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) [2]. Concerningly, an estimated 6.21 million middle and high school students reported trying any kind of tobacco product in the United States in 2022 [3]. Early use greatly increases the likelihood of developing dependence or escalation to more harmful substances [4,5,6]. Smoking and smoking-related diseases adversely impact individual healthcare and impose substantial economic burdens [7]. 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 [8,9,10,11,12]. The expression patterns and binding properties of nAChR subunits vary across brain regions but follow distinct developmental events in the cholinergic system [9,13,14]. The subunit configuration of these receptors regulates pharmacological properties such as agonist affinity/efficacy, desensitization, and downstream signaling, and is heavily implicated in presynaptic neurotransmitter release [10,15]. 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 [16,17,18,19]. Genome-wide association studies have also associated the α6 subunit gene, CHRNA6, with nicotine and other substance use [20,21,22,23,24]. The goal of this review is to evaluate advances in the study of the α6 subunit, its role in substance use disorders and other diseases, evaluate genetic influences on behavior, and discuss potential implications for the development of future therapeutic treatments.

2. Structure and Distribution of α6-Containing Neuronal nAChRs

2.1. Structure of Neuronal nAChRs

Neuronal nAChRs are ligand-gated ion channels found in the central and peripheral nervous systems [25]. The endogenous neurotransmitter acetylcholine (ACh) and exogenous ligands such as nicotine bind to these pentameric receptors, which are composed of five subunits [26]. In vertebrates, nAChRs exhibit diverse subunit compositions, with nine α (α2–α10) and three β subunits (β2–β4) identified [25,27,28]. Each of these subunits is made up of a long extracellular N-terminus, which is involved in ligand binding; four transmembrane domains (M1–M4); an intracellular cytoplasmic loop; and a short extracellular c-terminus [26,29] (Figure 1A). The second transmembrane domain (M2) of each subunit lines the permeable pore of the receptor, and the M1 and M3 transmembrane domains shield the M2 domain from the cell membrane. The M4 transmembrane domain, on the other hand, has the most contact with the cell membrane [11].

nAChR subunits can assemble into either heteromeric receptors containing at least one α and one β subunit (Figure 1C) or homomeric receptors in which all subunits are identical (Figure 1B). 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 [30,31]. 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 [25,32,33]. The specific arrangement of subunits within each receptor is highly regulated and dictates its unique pharmacology and function [34]. 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.

2.2. 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 [35,36,37,38,39,40,41,42]. 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 [18]. In the ventral striatum, about 60% of α6*nAChRs are composed of two α6β2 binding interfaces [18]. 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 [43]. Previous findings demonstrating α6 transcript expression with α4, β2, and β3 transcripts in roughly 90% of VTA dopaminergic neurons have been confirmed [8,9,36]. It has been 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 [43]. Notably, α6 subunit expression was previously believed to be restricted to VTA dopaminergic neurons [44]. However, recent work challenges that assumption by identifying α6 transcripts in roughly half of both GABAergic and glutamatergic neurons within the VTA [43,45,46], highlighting a broader distribution than previously recognized. While the α6 subunit had been previously identified in the LC of adolescent rats [35], the specific cell types expressing it were not determined. New findings now reveal α6 expression in GABAergic, glutamatergic, and noradrenergic neurons within the LC [43]. Furthermore, noradrenergic neurons may express α6(non-α4)β2β3 nAChRs, whereas GABAergic and glutamatergic neurons potentially express both α4α6β2β3 and α6β2 [43,47].

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 [48,49,50,51,52]. 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 [43]. Additionally, age- and sex-related differences in nAChR expression have been 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 [4,6,9]. 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 [53]. In MHb, α6 has been detected with β2 and β3, suggesting the receptor composition of α6β2β3 [40]. It has been suggested that combined exposure to high-dose nicotine and menthol may lead to a reduction in α6β2β3*nAChRs within this nucleus [53]. 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 [12,54].

Taken together, recent work has advanced our understanding of α6*nAChR expression patterns (Figure 2;

Table 1. A summary of the distribution of various α6*nAChRs in the brain.

α6*nAChR Subtype	Brain Region	Organism	Supporting Publication	Neuronal Population
α6β2	- Ventral and dorsal striatum - LC	Mouse Rat	[34] [55] [47] [18] [56]	Dopamine GABA Glutamate Norepinephrine
α6β4	- Striatum - LC - Hippocampus	Mouse	[56]	Dopamine Norepinephrine
α6β2β3	- Striatum - Medial habenula - Superior colliculus - Lateral geniculate nucleus	Mouse	[34] [55] [40] [57] [38]	Dopamine Norepinephrine
α6β3β4	- Interpeduncular nucleus	Mouse	[58]	-
α4α6β2	- Striatum - VTA	Mouse	[18] [56]	Dopamine GABA
α4α6β2β3	- VTA - Retinal ganglion cells - Striatum - LC - Superior colliculus - Lateral geniculate nucleus	Mouse	[43] [34] [55] [39] [38]	Dopamine GABA Glutamate
α4α5α6β2β3	- VTA	Mouse	[43]	Dopamine

). 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.

3. Conventional and Novel Strategies in Studying α6

While homomeric α7 and heteromeric receptors containing α4 and β2 subunits have been extensively studied [30], 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 [59]. The high sequence homology between the α6 and α3 subunits (61% amino acid similarity) further complicates research efforts [60]. Additionally, the absence of a specific antibody for the α6 subunit has hindered investigations into its expression patterns [61]. 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 [56,62]. 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 [63], 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 [64], leading to insights into the involvement of α6β2* and α4α6β2* receptors in striatal and NAc dopamine release [57,65,66]. Furthermore, α-contoxin PIA (α-CntxPIA) is the first conotoxin to distinguish between α6* and non-α6* nAChRs [67,68]. 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 [69]. α-Cntxs have been used widely across both in vitro and in vivo studies exploring α6*nAChRs. While the use of α-Cntxs offers substantial mechanistic insight, the 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 [70]. The following sections explore other methods of investigating α6*nAChRs and discuss developing novel strategies.

3.1. 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 the 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 [71]. 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 [72,73]. As part of efforts to investigate the functional expression of these receptors, it has been established that 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 [74]. However, these receptors remain intracellular even with NACHO, suggesting that additional factors are required for their functional expression [74,75]. 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 [76]. 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 [77]. 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 [77]. 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.

3.2. 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 has demonstrated the role of α6*nAChRs in nicotine-induced dopamine release and self-administration [8,16]. The α6-GFP mouse model, where there is a GFP fused in-frame into the M3–M4 intracellular loop of the subunit, has been a valuable tool that allows for high-resolution visualization of α6*nAChRs in the mouse visual system [39]. Given the broader expression of α6*nAChRs, including in astrocytes [43], 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) is mutated to serine by homologous recombination [17]. 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 [17]. This model is particularly promising for investigating locomotor activity and dopamine release in response to novel α6*nAChR agonists and antagonists. Such innovative approaches have also been applied to other nAChR subunits [78,79,80,81].

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, is associated with enhanced smoking behavior in humans (

Table 2. Clinical studies of the rs2304297 SNP, located in the 3′ UTR of CHRNA6, associated with nicotine/tobacco use.

Supporting Publication	Adolescent	Sample Size	% Male	“G” as Risk Allele
[27]	Yes	480	40	Yes
[83]	No	12,507	40.9	No %
[23]	Yes	439	41	Yes
[84]	No	6178	48	No %
[85]	Yes	1293	35	No #
[86]	No	789	48.9	Yes *
[87]	No	76,681	Not Reported	Yes *
[22]	Yes	423	45.4	Yes
[21]	Yes	1051	49.2	Yes
[21]	Yes	1051	49	No +
[20]	Yes	2674	48	Yes
[88]	Yes	1056	58.1	Yes
[89]	No	1929	38	Yes
[90]	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.

). To investigate the role of this SNP, a novel transgenic animal model was developed by replacing a rat CHRNA6 3′ UTR with the human version, creating two “humanized” rat lines: one homozygous GG (α6^GG) and one homozygous CC (α6^CC) [82]. 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.

3.3. Computational Methods

The utilization of lead compounds and computational modeling represents a well-established approach to 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 [91], 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 [92]. 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.

3.4. 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 [59,61]. 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 [93]. 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 [93]. 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 [94,95]. 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 [96], 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.

4. 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 [26]. These receptors play crucial roles in both excitatory and inhibitory neurotransmission [29], impacting cognitive function, synaptic plasticity, learning and memory, and reward pathways [28,97,98,99,100]. α6*nAChRs, particularly within the dopaminergic neurons of the mesolimbic pathway, have a major role in modulating dopamine release in the striatum [17]. Dopamine activity within the mesolimbic pathway is highly implicated in drug response and the development of drug dependence [101]. 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 [17,55,102]. These receptors are associated with nicotine-induced dopamine release and locomotor activity in rats [17,35], as well as nicotine self-administration [16,18,103]. 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 [104]. Neuroplasticity and synapse formation are other critical functions involved in substance use and dependence [93,105], with important implications for age- and sex-related differences in maturation of the dopamine system and drug response [9]. 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.

4.1. Nicotine

4.1.1. A: α6*nAChRs and Dopamine Release

Nicotine exerts its rewarding effects by acting on nAChRs within the mesolimbic dopamine pathway [106]. 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 [107] (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 [36,108], have been shown to modulate nicotine-induced dopamine release, particularly in the ventral striatal NAc compared to the dorsal striatal caudate putamen [66]. Additionally, intra-VTA perfusion of α-CntxMII, selective for α3/α6β2, and α-CntxPIA, selective for α6β2, suppresses nicotine-induced dopamine release in the NAc [18,109]. The VTA region can be divided into three subregions: anterior VTA (aVTA), posterior VTA (pVTA), and tail VTA (tVTA) [109]. 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 [109]. 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 [109]. Further studies in knockout and wild-type mice demonstrated the importance of both α4 and α6 subunits in the modulation of dopaminergic neurons and suggest that these effects are mediated by α6α4β2*nAChRs in the pVTA [18,109,110].

4.1.2. B: α6*nAChRs and Locomotor Activity

One of the behavioral outputs of dopamine release in rodents is locomotor activity [111]. Oligonucleotides against the α6 subunit result in partial inhibition of nicotine-induced locomotion, suggesting the involvement of α6*nAChRs [35]. The highly specific α-CntxPIA infused into the pVTA inhibits nicotine-induced locomotion and dopamine release [18]. 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 [12,17]. The hypersensitive α6^L9′S mouse line was used to demonstrate increased baseline locomotion compared to wild-type mice [17]. 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 [41]. Administration of the α6β2 antagonist, N, N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), to male and female mice induced a decrease in both locomotor activity in females between 10 to 30 min post-injection [112]. Using protein kinase C epsilon (PKCε) knockout female mice, enhanced nicotine-induced locomotor activity was associated with increased α6 and β3 expression [12]. PKCε is a critical signaling molecule that regulates the CHRNA6-CHRNB3 cluster [86] 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 [113]. 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 in VTA dopaminergic neurons.

4.1.3. C: 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 [16]. 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 the blockade of these receptors reduced rats’ motivation for reinforcement [103]. Moreover, pretreatment with α-CntxMII in the VTA, blocking α6*nAChRs, decreased nicotine self-administration response in rats [18]. Systemic administration of the α6β2 antagonist, bPiDI, also impairs acquisition and maintenance of nicotine self-administration [114,115]. 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.

4.1.4. D: Clinical and Pre-Clinical Studies Investigating CHRNA6 Encoding for the α6 Subunit

Clinical research has established a strong genetic component to nicotine dependence, with heritability estimates ranging from 40–70% [116,117,118]. 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 an SNP in the coding region of the CHRNA5 gene, encoding the α5 nAChR subunit, has been previously studied and linked to nicotine dependence [119,120], 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 [121]. For CHRNA6, rs2304297, an SNP in the 3′ UTR of the gene has been found to be associated with nicotine dependence in Caucasians [21,89]. There is an abundance of evidence associating this SNP with nicotine-seeking behaviors and susceptibility to nicotine addiction in humans, particularly adolescents (Table 2) [22,87]. 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 an increased positive perception of nicotine during adolescence and an increase in daily cigarette use (Table 2) [85,88].

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 rat CHRNA6 gene 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 [82]. Furthermore, adolescent male α6^GG rats were more prone than male α6^CC rats to relapse into nicotine-seeking behavior after drug plus cue exposure [122]. 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 [123] (

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	1. Increased locomotor activity and anxiolytic behavior in nicotine-treated male α6GG rats compared to saline controls 2. Increased anxiolytic behavior in nicotine-treated α6GG males compared to α6CC males	Increased locomotor activity and anxiolytic behavior in nicotine-treated α6CC rats compared to saline controls as well as nicotine-treated α6GG rats
Dopamine Release	Increased dopamine overflow in response to nicotine and methamphetamine in α6GG mice compared to α6CC rats	Increased dopamine overflow in response to nicotine and methamphetamine in α6CC rats compared to α6GG rats
Methamphetamine Self-administration	No genotype effect observed in methamphetamine self-administration after exposure to nicotine	Enhanced discrimination between reinforced and non-reinforced response in nicotine-treated α6CC rats and saline-treated α6GG rats
Tissue Neurotransmitter Levels	1. Nicotine + cue-treated adolescent α6GG rats had decreased dopamine levels in the NAc shell compared to other groups 2. Baseline differences: drug-naïve adolescent α6GG rats had greater dopamine levels in the NAc core compared to their adult counterparts and adolescent α6CC rats	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 [123] (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 [123]. 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 into novel mechanisms in nicotine-induced behaviors driven by an SNP in a 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.

4.2. Alcohol

The frequent co-use of alcohol and tobacco in humans is well established [124,125,126]. Indeed, individuals with alcohol use disorder (AUD) often have nicotine dependence [127,128]. Bidirectional associations between nicotine and alcohol have been described across several studies [129,130]. 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 [131]. Human genetic variations in CHRNA6, encoding for the α6 subunit, have been linked to smoking and alcohol in some instances (Table 2) and support 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 [132]. Injection of mecamylamine, a non-selective nAChR antagonist, into mouse VTA attenuates ethanol-induced increases in dopamine levels in the NAc [133,134,135]. nAChRs are also associated with alcohol-related behaviors such as sedation [136], locomotor activity [112,134], and intake [112,137]. Specifically, α6*nAChRs are necessary for ethanol-induced activation of VTA dopamine neurons and increased ACh release in mouse tissue slices [134,138]. 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 [55]. Transgenic mice with heightened nicotine sensitivity, α6L9′S, also show enhanced ethanol sensitivity, alcohol intake and conditioned place preference [139], suggesting a role for the α6 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 [55,139,140]. 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 a clear overlap in α6 subunit involvement for both nicotine- and alcohol-related dopamine function, a growing body of literature strongly implicates the regional and circuit-level influence of GABA [45,46,55]. α6*nAChRs are expressed on GABAergic terminals projecting onto both dopamine and GABAergic neurons within the VTA [43,45,141]. 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 [45,142] but inhibit these effects at high ethanol doses [55]. 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 [45,46,55].

While α6*nAChRs are strongly implicated in mediating ethanol’s rewarding effects, the precise mechanisms by which high doses of ethanol inhibit 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.

4.3. 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 [25,57,143,144]. 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 [145,146]. 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 motor symptoms [147] (Figure 4). L-DOPA crosses the blood–brain barrier and is converted to dopamine, whereas carbidopa inhibits DOPA decarboxylase and prevents its metabolism [147]. This treatment can lead to L-DOPA-induced dyskinesia (LID) within months of starting therapy [148,149]. 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 [150,151]. 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 [151,152].

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 [153,154,155,156]. In the 6-hydroxydopamine-induced nigrostriatal lesion model of PD, both α4β2*- and α6β2*-mediated dopamine release is progressively lost with increased nigrostriatal damage [146]. 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 [157] (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 [140]. In this case, AN6001 exhibits significantly higher selectivity for α6β2* compared to α4β2* nAChRs, one of the most abundant nAChRs in the brain [157]. 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.

4.4. 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) [158,159,160]. P2X2/3 receptors are ionotropic receptors that are involved in the development of neuropathic pain [160]. 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 [161,162,163]. 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 [164]. The immunohistochemistry of mouse DRG α6-containing neurons has also shown nociceptive neuron markers [165]. 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 [165]. 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 [165]. Additionally, the antiallodynic effect of nicotine has been investigated using α4 and α6 KO as well as wild-type mice [165]. 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 [165]. 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 [166]. In vivo testing of ABT-594 in BARP-KO mice which had reduced expression of α6β4 has shown blunted antiallodynic effects as compared to wild-type controls [166]. 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 [163], highlight the complexities of translating preclinical findings to clinical applications.

Genetic variations within the CHRNA6 gene have been studied in pain clinical studies [165]. More specifically, a rs7828365 SNP within the promoter region has been 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. While 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 [165]. 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.

5. Conclusions

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 the 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 [59]. 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 the basic mechanisms of action for substances and the 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 [43,45,46]. 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 [106,107,131].

The adolescent brain is especially susceptible to the effects of nicotine [6], 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 [166], and adolescent females show increased sensitivity to its rewarding effects [167]. α6 subunit mRNA expression peaks in VTA and substantia nigra dopamine neurons during adolescence in male rats [108]; 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 [43,53]. As reviewed in Meon et al. (2021) [12], there are clear sex differences in nAChR expression patterns throughout the brain in adults with various implications for 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 [87,168,169]. Common genetic factors underlying nicotine and alcohol codependency are important to evaluate since they 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 [151]. 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.