Expression of cloned α6* nicotinic acetylcholine receptors

Neuropharmacology 96 (2015) 194e204

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Invited review

Expression of cloned a6* nicotinic acetylcholine receptors Jingyi Wang, Alexander Kuryatov, Jon Lindstrom* Department of Neuroscience, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 16 October 2014

Nicotinic acetylcholine receptors (AChRs) are ACh-gated ion channels formed from five homologous subunits in subtypes defined by their subunit composition and stoichiometry. Some subtypes readily produce functional AChRs in Xenopus oocytes and transfected cell lines. a6b2b3* AChRs (subtypes formed from these subunits and perhaps others) are not easily expressed. This may be because the types of neurons in which they are expressed (typically dopaminergic neurons) have unique chaperones for assembling a6b2b3* AChRs, especially in the presence of the other AChR subtypes. Because these relatively minor brain AChR subtypes are of major importance in addiction to nicotine, it is important for drug development as well as investigation of their functional properties to be able to efficiently express human a6b2b3* AChRs. We review the issues and progress in expressing a6* AChRs. This article is part of the Special Issue entitled ‘The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition’. © 2014 Published by Elsevier Ltd.

Keywords: a6 Nicotinic acetylcholine receptor Expression Assembly Nicotine

1. Introduction

a6b2b3* AChRs are of special neuropharmacological interest for several reasons. a6, a4, and b2 subunits are required to form AChRs critical for addiction to nicotine, because knockout of any of these subunits prevents nicotine self-administration in mice (Pons et al., 2008). AChRs assembled from these subunits (i.e., a6a4b2* subtypes) are identified by immune-isolation and study of knockout mice in midbrain dopaminergic neurons, which are critical for nicotine reward and addiction (Champtiaux et al., 2003; Drenan et al., 2010; Gotti et al., 2010; De Biasi and Dani, 2011). b3 subunits are adjacent to a6 subunits in the genome and they are usually co-expressed (Han et al., 2000; Quik et al., 2000; Cui et al., 2003). This suggests that the complex (a6b2)(a4b2)b3 AChR subtype is important for nicotine addiction. This subtype in dopaminergic nerve endings promotes release of dopamine (Salminen et al., 2007; Drenan et al., 2010; Exley et al., 2011; Liu et al., 2012). It is the subtype controlling dopamine release that is most sensitive to activation by nicotine (Salminen et al., 2007; Kuryatov and Lindstrom, 2011). Loss of Abbreviations: ACh, acetylcholine; AChR, nicotinic acetylcholine receptor; a-Ctx, a-conotoxin; eGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GABA, gamma-aminobutyric acid; HEK, human embryonic kidney; N-2a, Neuroblastoma 2a; SNc, substantia nigra pars compacta; VTA, ventral tegmental area. * Corresponding author. Department of Neuroscience, 217 Stemmler Hall, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 191046074, USA. Tel.: þ1 215 573 2859. E-mail address: [email protected] (J. Lindstrom). http://dx.doi.org/10.1016/j.neuropharm.2014.10.009 0028-3908/© 2014 Published by Elsevier Ltd.

a6* AChRs is an early sign of dopaminergic neuron loss in Parkinson's disease (Gotti et al., 2006; Quik et al., 2011; Srinivasan et al., 2014). Both (a6b2)(a4b2)b3 and (a6b2)2b3 subtypes of AChRs are vulnerable to nigrostriatal damage in an animal model of Parkinson's disease (Quik et al., 2005). Transgenic mice expressing a hypersensitive form of a6 subunit exhibit enhanced dopaminerigic neuron activity and locomotor hyperactivity (Drenan et al., 2008, 2010). Each AChR has five homologous subunits: a6 and b2 form one acetylcholine (ACh) binding site, a4 and b2 form another ACh binding site, and b3 is the accessory subunit (Millar and Gotti, 2009; Hurst et al., 2013). Having two types of ACh binding sites in (a6b2)(a4b2)b3 AChRs suggests that they have unusual neuropharmacological properties. a-Conotoxin MII (a-CtxMII) is an antagonist for ACh binding sites formed at the interface of a6 and b2 subunits, and a critical tool for localizing and identifying the function of a6b2* AChRs (Whiteaker et al., 2000; Champtiaux et al., 2003; McIntosh et al., 2004). a-CtxMII is an antagonist for both a6b2* and a3b2* AChRs, but is often used for detecting a6b2* AChRs because in brain the a3 subunit is expressed almost exclusively in the medial habenula (Cartier et al., 1996; Kuryatov et al., 2000; Han et al., 2000; Whiteaker et al., 2002; McIntosh et al., 2004). Several a-conotoxin variants that are more selective for a6b2* AChRs were subsequently discovered (Dowell et al., 2003; McIntosh et al., 2004; Azam et al., 2010). As peptides, these a-conotoxins cannot cross the bloodebrain barrier, or lead to development of small molecule therapeutics targeting a6* AChR related neurological disorders.

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In order to study the neuropharmacology of a6* AChRs, understand their pharmacology, and develop bioavailable drugs specific for them, it is necessary to express specific human subtypes of these AChRs. Neuronal cell lines for this purpose are not available. Expressing cloned AChRs has challenges. Although functional expression of AChRs, including a6b4* AChR subtypes, is generally easy to achieve in the Xenopus oocyte system (Gerzanich et al., 1997; Kuryatov et al., 2000; Broadbent et al., 2006; Dash et al., 2011a), expression of a6 and b2 forms many (a6b2) ACh binding sites that can be labeled with 3H-epibatidine but not mature functional AChRs on the oocyte surface (Gerzanich et al., 1997; Kuryatov et al., 2000). Expressing a6b2* AChRs in cultured cell lines is even more difficult. Human a6* AChRs can be expressed in transfected HEK cells, and b3 can increase their sensitivity to up-regulation by nicotine, but the level of expression for both a6b2b3 and a6b4b3 AChRs is too low for assaying AChR function even after nicotine up-regulation (Tumkosit et al., 2006). (a4b2)2b3 AChRs assemble very efficiently in transfected HEK cells to form functional AChRs (Kuryatov et al., 2008). However, transfection of an a4b2 HEK cell line with a6 does not result in efficient assembly of a6a4b2 AChRs (Kuryatov et al., unpublished). Another issue of expressing heteromeric AChRs in both oocytes and cells is the potential of forming multiple stoichiometries with distinct properties, such as (a4b2)2b2 and (a4b2)2a4 (Zwart and Vijverberg, 1998; Nelson et al., 2003; Kuryatov et al., 2005; Sallette et al., 2005; Harpsøe et al., 2011; Mazzaferro et al., 2011). These challenges are being overcome through the use of mutants, chimeras, and concatamers (Kuryatov et al., 2000, 2011; Broadbent et al., 2006; Capelli et al., 2011; Jensen et al., 2013, 2014; Henderson et al., 2014; Ley et al., 2014). This review focuses primarily on the significance and expression of a6b2* AChRs, but compares them with what is known about a6b4* AChRs.

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2. Neuropharmacological properties of a6b2b3* AChRs One of the gold standards to locate and distinguish the neuropharmacological properties of various AChR subtypes is the use of selective ligands, such as a-bungarotoxin for a7 and muscle type AChRs or DhbE or epibatidine for b2-containing AChRs (Hurst et al., 2013). A 16 amino acid peptide, a-conotoxin MII (a-CtxMII) from the marine cone snail Conus magus, was initially found to be a highly selective antagonist for a3b2* AChRs, and later identified to also have high affinity for a6b2* AChRs, but very low affinity for a2* and a4* AChRs (Cartier et al., 1996; Kuryatov et al., 2000; McIntosh et al., 2004). This toxin subsequently become a useful tool for identifying a6b2* AChRs and evaluating their importance in the effects of nicotine both in brain and in heterologous systems as described here and in Sections 3.1.1 and 4.1. In 2000, Whiteaker et al. developed a radioactive version of this toxin, 125I a-CtxMII, which allowed locating a6* AChR subtypes in brain tissue. Homozygous null mutant (a6/) mice showed complete loss of brain [125I] a-CtxMII binding sites (Champtiaux et al., 2002), and a3 knockout mice showed no significant loss of [125I] a-CtxMII binding sites, except in the habenulointerpeduncular nuclei (Whiteaker et al., 2002). This suggests that a6, rather than a3, is critical for dopamine release in brain. The percentage and sensitivity of a6b2* AChR subtypes are obtained by assessing the portion of agonist-stimulated release of dopamine which is sensitive to a-CtxMII block, as discussed in Section 3.1.1 and 3.2 (Table 1). Subsequently, various a-conotoxins and their mutants were developed with equal or better selectivity for a6 versus a3 than that of a-CtxMII, and used to identify a6* subtypes and their physiological importance in aminergic neurons (Dowell et al., 2003; McIntosh et al., 2004; Azam et al., 2005, 2010; Luo et al., 2013). Via analyzing sequences interacting with AChRs, a mutant form of

Table 1 Functional characterization of a6b2* AChRs in dopaminergic neurons. Subtypes

Species

Location

EC50 (mM)

a6* AChRs (% of

Reference

total response)

a6b2*

Mouse

Monkey

a6L90 Sb2*

a6(non a4)b2*

Mouse

Mouse

a6L9 S(non a4)b2*

Mouse

a6(non b3)* a6(non a4 or b3)* a6a4b2b3 a6b2b3

Mouse Mouse Mouse

0

Striatal synaptosomes Striatal synaptosomes Striatal synaptosomes Olfactory tubercle Striatal synaptosomes Striatum Olfactory tubercle Dorsal striatum Olfactory tubercle Caudate Putamen Nucleus accumbens Nucleus accumbens Striatal synaptosomes

0.77 0.81 0.11 0.082 0.62 0.099 0.110 0.031 0.075 0.31 0.32 0.58 0.33 0.93

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.27 0.12 0.04 0.037 0.19 0.026 0.024 0.017 0.025 0.10 0.10 0.24 0.16 0.13

33 26 26 21 29 15 15 17 24 68 70 80 75 80

Striatal synaptosomes Olfactory tubercle Dorsal striatum Olfactory tubercle

0.047 0.025 0.016 0.015

± ± ± ±

0.011 0.004 0.006 0.004

58 65 46 67

Dorsal striatum Olfactory tubercle Striatal synaptosomes

0.88 ± 0.16 0.97 ± 0.15 0.103 ± 0.031

Dorsal striatum Olfactory tubercle Striatal synaptosomes Striatal synaptosomes Striatal synaptosomes

0.25 0.43 0.42 21.24 0.23 1.52

± ± ± ± ± ±

0.02 0.09 0.83 3.21 0.08 0.19

Salminen et al., 2004 Cui et al., 2003 Drenan et al., 2008 Salminen et al., 2007 Marks et al., 2014 Drenan et al., 2010 McCallum et al., 2005

McCallum et al., 2006a Perez et al., 2012 Drenan et al., 2008 Drenan et al., 2010 Drenan et al., 2010 Champtiaux et al., 2003 Drenan et al., 2010

5.1 4.6

Cui et al., 2003 Salminen et al., 2007 Salminen et al., 2007

Function was assayed by measuring [3H]-dopamine release induced by nicotine. Percent of total response contributed by a6* AChRs was assayed using the a6-selective antagonist, a-Ctx MII. a6L90 S: a6 gain-of-function mutant.

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a-CtxMII[E11A] was developed that exhibited more than 50 fold higher binding affinity for a6b2* AChRs than a3b2* AChRs

a6b2, a6b4, a6b2b3, a6a4b2, a6b2b4, a6b4b3, a6a4b2b3, and a6a3b2b3 (Fig. 1; Champtiaux et al., 2003; Gotti et al., 2005a,b,

(McIntosh et al., 2004). This E11A mutant was subsequently used to identify a6b2* AChRs and their neuropharmacological roles in various neurons (Perez et al., 2008; Hone et al., 2012; Liu et al., 2013). Similarly, a mutated form a-CtxBuIA[T5A; P6O] was generated with selectivity for a6b4* AChRs versus a6b2* AChRs, which led to identification of both subtypes in hippocampus (Azam et al., 2010). Microinfusion of a-CtxMII in the nucleus accumbens shell or the ventral tegmental area reduces nicotine self-administration by rats (Brunzell et al., 2010; Gotti et al., 2010). Injection of an a6-selective a-Ctx variant to mouse brain attenuates both reward and withdrawal effects of nicotine (Jackson et al., 2009; Sanjakdar et al., in press) and cocaine (Sanjakdar et al., in press). However, because a-conotoxins are peptides, they do not provide leads to small molecule drugs.

2010; Salminen et al., 2004, 2005, 2007; Grady et al., 2002, 2007, 2009; Moretti et al., 2004; Cox et al., 2008; Azam et al., 2010; Beiranvand et al., in press). Therefore, it is important to understand locations, subtypes, and pharmacological properties of a6* AChRs in vivo to guide and validate the expression of cloned a6* AChRs using mutants, chimeras and concatamers described in Sections 4.1 and 4.2. a6 subunit mRNA is expressed prominently in mid-brain dopaminergic neurons, retina, visual nuclei, locus coeruleus and medial habenula of rodents and a non-human primate (Le Novere et al., 1996; Quik et al., 2000; Han et al., 2000; Moretti et al., 2004). [125I] a-CtxMII and a6 specific antibodies confirm expression of a6* AChRs in these areas in both humans and other mammals (Whiteaker et al., 2000; Champtiaux et al., 2002; Cox et al., 2008; McCallum et al., 2005; Gotti et al., 2005b, 2006). a6* AChRs were also recently identified in hippocampus and some peripheral tissues using an a-conotoxin (Azam et al., 2010; Hone et al., 2012;
rez-Alvarez et al., 2012; Herna
ndez-Vivanco et al., 2014). The Pe constitution and neuropharmacological function of these a6* AChRs will be discussed in the following sections.

3. Expression of a6* AChRs in animals Even though a6* AChRs are minor subtypes in brain, they form various simple and complex subtypes including, but not limited to,

3.1. Identification and composition of a6* AChRs in dopaminergic neurons and the visual system

Fig. 1. Subtypes and locations of a6* AChRs in vivo. a6* AChRs are minor but important subtypes of AChRs. a6 subunits are primarily associated with b2 subunits in the brain, as determined by immunoisolation (Champtiaux et al., 2003; Gotti et al., 2005b) and b2-null mice (Salminen et al., 2005; Grady et al., 2007). These a6b2* AChRs are prominently expressed in midbrain dopaminergic neurons and retinal ganglionic neuron termini where they modulate dopamine release (Grady et al., 2002; Salminen et al., 2004; Gotti et al., 2005b). They are also present in retina, habenula, hippocampus, and etc. (Le Novere et al., 1996; Moretti et al., 2004; Marritt et al., 2005; Grady et al., 2009; Azam et al., 2010; Beiranvand et al., in press). In dopaminergic neurons, (a6b2)2b3 and (a6b2)(a4b2)2b3 subtypes contribute to more than 70% of a6* AChRs (Gotti et al., 2010); they differ in the second ACh binding site (i.e. a6/b2 versus a4/b2 interface), thus showing distinct sensitivities to nicotine (Salminen et al., 2007). a6b4* AChRs are less prevalent than a6b2* AChRs in the brain (Azam et al., 2002; Cui et al., 2003), but are present in hippocampus and some peripheral tissues (Azam et al., 2010;
rez-Alvarez et al., 2012; Hern
andez-Vivanco et al., 2014). a3a6* Hone et al., 2012; Pe AChRs are a small population of a6* AChRs (<16%) in retina and optic nerves (Moretti et al., 2004; Marritt et al., 2005; Cox et al., 2008).

3.1.1. Immunoisolation and binding of a-conotoxins a6b2* AChRs are most densely expressed in visual nuclei (Whiteaker et al., 2000; Champtiaux et al., 2002), but most extensively studied in dopaminergic neurons, because of their important presynaptic roles in modulating dopamine release in nicotine addiction (Table 1). a6b2* AChRs are expressed in two major dopamine systems, the mesolimbic pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens and the tuberculum olfactorium, which are important for reward, learning, and addiction (McCallum et al., 2005; Exley et al., 2008, 2011; Pons et al., 2008; Liu et al., 2012; De Biasi and Dani, 2011), and the nigrostriatal pathway projecting from the substantia nigra pars compacta (SNc) to the dorsal striatum (i.e., caudate-putamen components), which are important for motor control (Zoli et al., 2002; Quik et al., 2005, 2011; Exley et al., 2013; Srinivasan et al., 2014). Non-human primates have a higher percentage of a6b2* AChRs than rodents (Table 1). Blockage of dopamine release by a-CtxMII in monkeys is about 75%, much higher compared to ~35% found in rodents in the striatum and the nucleus accumbens (Kulak et al., 1997; Quik et al., 2003; Salminen et al., 2004; McCallum et al., 2005, 2006a). Thus, a6b2* AChR drugs may be even more important in humans than their effects in rodents would suggest. This emphasizes the importance of cloning and expressing human a6b2b3* AChRs. In brain dopaminergic systems, b2 plays an essential role in all a6* AChR subtypes because b2 mRNA, rather than b4, is predominantly expressed in these areas (Fig. 1; Azam et al., 2002; Cui et al., 2003). In AChRs immunoisolated from dopaminergic neurons, b2 subunits are in most a6* AChRs (Champtiaux et al., 2003; Gotti et al., 2005b). Knocking down the b2 gene abolishes [125I] aCtxMII binding in dopaminergic terminals (Salminen et al., 2005; Grady et al., 2007; Baddick and Marks, 2011) as well as nicotineinduced dopamine release that is sensitive to a-CtxMII block (Grady et al., 2002; Salminen et al., 2004). The b3 subunit is in most a6b2* AChRs. Knockout of the b3 gene in mice dramatically reduces maximum a-CtxMII-sensitive release of [3H]-dopamine and expression of a6* AChRs in striatal synaptosomes (Table 1; Cui et al., 2003; Gotti et al., 2005a,b; Salminen

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et al., 2005, 2007; Baddick and Marks, 2011). b3 increases sensitivity of a6* AChRs to nicotine up-regulation (Tumkosit et al., 2006). Immunoisolation studies have determined that more than 70% of brain a6* AChRs contain b3 subunits, except in optic nerves (Gotti et al., 2005b, 2010; Cox et al., 2008). Some a6* AChRs contain a4 subunits (Fig. 1). Immunoprecipitation and a4 knockout mice identified two major populations of a6b2* AChRs sensitive to blockage by a-CtxMII in dopaminergic neuron terminals, i.e., a6a4b2* and a6b2* AChRs (Zoli et al., 2002; Champtiaux et al., 2003; Salminen et al., 2005, 2007; Grady et al., 2007; Gotti et al., 2010; Baddick and Marks, 2011; Exley et al., 2011; Liu et al., 2012). It is important to express and study physiological properties of these a6* AChR subtypes with defined subunit compositions. a6a4b2* and a6b2* AChRs are expressed in different ratios in rat mesolimbic and nigrostriatal neurons, as deduced from immunoprecipitation experiments (Gotti et al., 2010). The a6b2b3 subtype accounts for 60% of a6* AChRs expressed in ventral striatum, while the a6a4b2b3 subtype dominates (89%) in dorsal striatum (Gotti et al., 2010). Such subtype heterogeneity suggests different neuropharmacological functions for various a6* AChR subtypes in vivo. A small portion of a6* AChRs (13e16%) is associated with a3 in the retina and optic nerves in rodents (Moretti et al., 2004; Cox et al., 2008). a6, a3, and b3 subunit mRNAs are preferentially concentrated in the rat retina ganglion cell layer (Moretti et al., 2004). Their protein level increases after birth until postnatal day 21, about a week after eye opening. In retina, the majority (>80%) of a6* AChRs contain b2 subunits, and nearly all immunoisolated a6a3* AChRs contain the b3 subunit (Moretti et al., 2004; Marritt et al., 2005). In optic nerves, b2 subunits contribute to all agonist binding sites in a6a3* and a6a3a4* AChRs (Cox et al., 2008). Removing eyes attenuates or abolishes expression of a6, a3, a4 and b3 subunits in retinal terminal areas (Gotti et al., 2005b). a-CtxMII specific for a6b2* and a3b2* AChRs blocked retinal waves present before eye opening (Bansal et al., 2000). Knockout studies have identified involvement of primarily b2, but also a3 and b4, AChR subunits in development of retinofugal projections (Bansal et al., 2000; Rossi et al., 2001; Grubb et al., 2003; Stafford et al., 2009; Dhande et al., 2011). Therefore, it is likely that a6b2*, a6a3b2*, a6a4b2*, and a6a3a4b2* AChRs play important roles in visual development and signal processing. Expressing these complex a6* AChRs from cloned subunits will help determine their specific electrophysiological and neuropharmacolgocial functions. 3.1.2. Genetic manipulation of a6* AChRs in mice Genetic manipulations of a6 in mice (e.g., preventing a6 expression or replacing it with hyperactive or labeled derivatives) provide another way to locate and understand the neuropharmacological properties of a6b2b3* AChRs. Genetic deletion of a6 subunits abolished [125I] a-CtxMII binding in both mid-brain dopamergic neurons and the visual system (Champtiaux et al., 2002), abolished [3H]-epibatidine binding of immunoprecipitated a6* AChRs (Gotti et al., 2005b), reduced nicotine self-administration (Pons et al., 2008), and obliterated conditioned place preference for cocaine (Sanjakdar et al., in press). a6-null mice developed normally, and did not show obvious behavior deficits (Champtiaux et al., 2002). These observations suggest that a6 antagonists targeting these minor AChR subtypes in the brain as a therapeutic for smoking cessation would have minimal side effects (Brunzell et al., in press; Crooks et al., 2014). Interpretation of a6 functional roles from knockout mice might be misleading if developmental alteration from gene inactivation or functional compensation from other AChRs were to occur (Champtiaux and Changeux, 2004). There is no change of mRNA levels of other AChR subunits in a6null mice (Champtiaux et al., 2002). There is no increased amount

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of other AChR subtypes in visual nuclei (Champtiaux et al., 2002; Gotti et al., 2005b), but a small (<18%) increase of a-CtxMII insensitive AChRs in striatum (Champtiaux et al., 2002). Therefore, normality of a6-null mice is not likely a result solely from compensations from other AChRs. a6 AChR antagonist therapeutics are promising for treating nicotine addiction. Genetic knockin of mutant a6 in mice provides an alternative approach, but with a different set of caveats due to possible effects of a6* AChRs with altered functions. Mice with gain-of-function 0 a6L9 S mutants showed locomotor hyperactivity and failed to habituate to a novel environment, due to increased dopamine release by activating a6b2* AChRs, especially a6a4b2* subtypes in dorsal striatum (Drenan et al., 2008, 2010). These hyperactive mice also consumed more alcohol, probably due to the reinforcing effect of a6* AChRs in VTA dopaminergic neurons (Powers et al., 2013). Enhanced synthesis of dopamine was observed in these hyperactive transgenic mice, which, together with increase of dopamine release, probably resulted in increased locomotion and drug addiction in these animal models (Wang et al., 2014). Both knockin and knockout studies suggest a6* AChRs as important targets for developing therapeutics for addictions to nicotine, cocaine and alcohol, and perhaps also Parkinson's disease. Transgenic mice expressing a6 subunits fused with green fluorescent protein provide another tool to localize a6* AChRs (Mackey et al., 2012). This localized labeled a6* AChR protein to retinal tissues as well as neurons in the VTA, the SNc, the medial habenula, and the superior colliculus (Mackey et al., 2012; Powers et al., 2013; Henderson et al., 2014). In the VTA and SNc, labeled a6 subunits were expressed in more than 88% of the catecholaminergic neurons and up-regulated after chronic nicotine exposure (Mackey et al., 2012; Henderson et al., 2014). 3.2. Neuropharmacology of a6* AChRs in dopaminergic neurons Most a6b2* AChR subtypes identified in transgenic mice and/or by a-CtxMII binding show high sensitivity to nicotine in releasing dopamine, as shown in Table 1. Incorporation of a4 in either wild type or hypersensitive a6b2* AChRs increases sensitivity of AChRs to nicotine, which differentiates (a6b2)(a4b2)b3 AChRs from (a6b2)2b3 AChRs (Salminen et al., 2007; Drenan et al., 2010). Upregulation of a6* AChRs by nicotine in brain is controversial (Srinivasan et al., 2014). Chronic nicotine treatment in vivo led to upregulation (Nguyen et al., 2003; Parker et al., 2004; Visanji et al., 2006; Perez et al., 2008; Henderson et al., 2014), or downregulation of a6*-containing AChRs in rodents and a non-human primate (Lai et al., 2005; McCallum et al., 2006b; Perry et al., 2007; Doura et al., 2008; Perez et al., 2008, 2012, 2013; Marks et al., 2014). In mouse brain, a6b2b3* AChRs are three-fold more sensitive to nicotine down-regulation than a4b2* AChRs are to nicotine up-regulation (Marks et al., 2014). Up-regulation of a6b2* AChRs by nicotine was observed in cell lines (see Section 4.3; Tumkosit et al., 2006; Henderson et al., 2014), while a4a6* AChRs were more vulnerable to down-regulation in the brain (Perry et al., 2007; Exley et al., 2013). Tumkosit et al. (2006) noted that upregulation of a6b2b3* AChRs was less sensitive to nicotine than was upregulation of a4b2* AChRs (Kuryatov et al., 2005) and suggested that in cells expressing both a6 and a4 low concentrations of nicotine would selectively upregulate a4b2* AChRs at the expense of a6b2* AChRs that would compete for the limited pool of b2 subunits available for assembly. In many brain areas a4 and b2 are in great excess over a6 subunits (Gotti et al., 2005a,b). If a4 and b2 were present in similar excess over a6 in a cell, then even partial upregulation of (a4b2)2b2 AChRs might significantly downregulate a6b2* AChRs, which could account for the report that sensitivity to nicotine concentration of downregulation of a6b2b3* AChRs is even

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higher than sensitivity to upregulation of a4b2* AChRs (Marks et al., 2014). This theory is also supported by evidence that the total number of specific radioactive ligands bound to a6b2* AChRs increases in a4-null mice after chronic nicotine treatment (Perez et al., 2008). Upregulation of AChRs is an issue of expression. We, and others, attribute upregulation by nicotine primarily to a pharmacological chaperone effect (Kuryatov et al., 2005; Sallette et al., 2005; Corringer et al., 2006; Srinivasan et al., 2014). Cholinergic ligand binding to partially assembled AChRs induces conformations that assemble more efficientlydthus nicotine binding to (a4b2)2 promotes assembly with b2 rather than a4 accessory subunits and increases the amount of the (a4b2)2b2 subtype (Kuryatov et al., 2005). We also found that nicotine slowed the rate of turnover of a4b2* AChRs in HEK cells (Kuryatov et al., 2005). Marks et al. (2011) observed increased a4 and b2 AChR protein in brains as a result of chronic nicotine exposure measured by using both subunit-specific antibodies and cholinergic ligand binding. Lester and other groups found increased a4 and a6 protein after nicotine upregulation detected in mice expressing fluorescently labeled a4 and a6 subunits (Renda and Nashmi, 2012; Henderson et al., 2014). 3.3. a6* AChRs in other brain areas and peripheral tissues The a6 AChR subunit is associated with b2, b4 and/or b3 subunits in other brain areas besides dopaminergic neurons and visual nuclei (Fig. 1). a6 and b3 mRNAs co-localize in habenula (Han et al., 2000; Quik et al., 2000). Transgenic mice express fluorescent a6* AChRs in the medial habenula (Henderson et al., 2014). Knock out of b2 abolished expression of a6* AChRs in mouse habenula and interpeduncular nuclei (Grady et al., 2009; Beiranvand et al., in press), suggesting the presence of a6b2* AChRs in the habenulainterpeduncular pathway. a6b4* AChRs regulate norepinephrine release in mouse hippocampus together with a6b2* subtypes, as evidenced by an a-conotoxin selective for a6b4* AChRs (Azam et al., 2010). (a6b4)2b3 AChRs are found at low levels in the habenulointerpeduncular pathway, primarily in the interpeduncular nucleus (Grady et al., 2009). The presence of a6b4* AChRs was detected in peripheral tissues using a mutated a-CtxBuIA[T5A;P6O] selective for a6b4 AChRs (Azam et al., 2010). This toxin partially blocked ACh-induced currents in dorsal root ganglia neurons and chromaffin cells in monkey
rez-Alvarez et al., and human adrenal glands (Hone et al., 2012; Pe
ndez-Vivanco et al., 2014). In human chromaffin cells, 2012; Herna a6b4* AChRs play an important presynaptic role, as suggested by
rezmembrane capacitance changes caused by a-conotoxins (Pe Alvarez et al., 2012). a6, b2, b3, and b4 subunit mRNAs were also identified in bladder afferent neurons (Nandigama et al., 2013). Subunit compositions of a6* AChR subtypes in these peripheral tissues are not clear. 4. Expression of cloned a6* AChRs in Xenopus ooctyes and cell lines Unlike other AChR subtypes that are often readily expressed in heterologous expression systems, functional expression of wild type a6b2* AChR is hard to achieve, even two decades after the first report of a6 mRNA (Lamar et al., 1990; Gerzanich et al., 1997). However, expression of cloned a6* AChRs with various modifications has made progress and facilitated studying functions of these AChRs in vivo. For example, antagonism of a-CtxMII on a6* was first identified by expression of a6/a3 or a6/a4 chimeras with b2 or b4 in oocytes (Kuryatov et al., 2000). From then on, a-CtxMII and its variants have been used extensively to locate and study neuropharmacological functions of a6* AChRs in brain (see Section 2 and

3.1.1). Although a6b2* AChRs are more important and prevalent than a6b4* AChRs in vivo (see Section 3), a6b4* subtypes are easier to express in heterologous systems, thus better investigated in mammalian cell lines (Gerzanich et al., 1997; Fucile et al., 1998; Evans et al., 2003; Jensen et al., 2013, 2014). We will discuss both a6b2* and a6b4* AChRs to investigate the barriers to expressing cloned a6* AChRs.

4.1. Expression in Xenopus oocytes Functional a6* AChRs were first expressed in Xenopus oocytes using chick or rat a6 and human b4 (Gerzanich et al., 1997). Subsequently, functional human (a6b4)2b3 AChRs were expressed in oocytes (Kuryatov et al., 2000). The presence of b3 subunits increased the response of (a6b4)2b3 AChRs four fold compared to a6b4 AChRs (presumably a mixture of (a6b4)2b4 and (a6b4)2a6) stoichiometries). A few residues in the extracellular domain of b3 have proven to account for increased expression of human/rat hybrid a6b2b3 and a6b4b3 AChRs compared to their pure human or rat versions (Dash et al., 2014). Some of these residues are spatially close to the N-terminal helix and could increase expression of AChRs by affecting cleavage of signal peptides. Other residues are at the tip of C-loop and could facilitate assembly and maturation of a6* AChRs. However, a great excess of b3 decreases total function of human a6b4b3 AChRs (Broadbent et al., 2006). This is probably due to accumulation of dead end intermediates, as observed with expression of excess a5 and b3 accessory subunits in a4b2* AChRs (Kuryatov et al., 2008). It was initially difficult to express functional a6b2* AChR in oocytes (Gerzanich et al., 1997; Kuryatov et al., 2000; Broadbent et al., 2006). Subsequently, functional a6b2* AChRs were expressed in oocytes using subunit chimeras, point mutagenesis of a6 or b3 subunits, or concatameric subunits (Figs. 2 and 3; Table 2; Kuryatov et al., 2000; Broadbent et al., 2006; Dash and Lukas, 2012; Kuryatov and Lindstrom, 2011; Ley et al., 2014). The transmembrane domain and cytoplasmic domain of a6 are the main barriers to expressing a6b2* AChRs in oocytes. Chimeras with the extracellular domain of a6 and the rest of a3 or a4 (illustrated in Fig. 2) co-expressed with b2 were able to produce functional AChRs in oocytes (Kuryatov et al., 2000). These chimeras were sensitive to blockage by a-CtxMII (Kuryatov et al., 2000) and were subsequently used to improve expression of other a6* AChR subtypes in oocytes (Evans et al., 2003; Dash et al., 2011b; Jensen et al., 2013). Sequences from the large cytoplasmic domain or transmembrane domains of a6 attenuated, or abolished functions of chimeras with a3 or a4 AChRs (Papke et al., 2008; Kuryatov and Lindstrom, 2011). Two amino acids, methionine 211 and phenyanaline 223, at the beginning of the first transmembrane domain of a6 in chimeras with a3 impeded expression of a3b2 AChRs. Recently, we developed an a6 variant with its large cytoplasmic loop and position 211 replaced with sequences of a3, which expressed robust current with free b2 and b3 subunits in oocytes (Ley et al., 2014). AChRs expressed from this new a6 chimera exhibit the pharmacological properties expected for (a6b2)2b3 AChRs. a6* AChRs expressed with gain-of-function mutants exhibited more functional current in oocytes (Broadbent et al., 2006; Dash and Lukas, 2012). Mutation in either a6 or b3, enabled functional expression of human a6b4b3, but not a6b2b3 AChRs (Broadbent et al., 2006; Dash et al., 2011a, 2012). With two additional mutations at the extracellular domain of a6, Dash and Lukas (2012) were 0 able to detect function of human a6L9 Sb2b3 AChRs. However, these gain-of-function constructs exhibited spontaneous channel opening and did not exhibit the pharmacological properties of wild type a6* AChRs (Dash et al., 2011a).

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Fig. 2. Illustration of modifications in cloned a6b2b3* AChRs. A) Indicates the basic schematic topology of an AChR subunit: a large rigidly structured extracelluar domain, a transmembrane domain of four a-helices (M1e4), and a large loosely structured intracellular domain between M3 and M4. B) Depicts a6 chimeras used to express a6* AChRs in vitro, including chimeras of a6 with a3 or a4 (Kuryatov et al., 2000). The a6 sequences replaced by other subunits in chimeras are indicated by dotted arrows. Amino acid sequences of chimeras are numbered as corresponding subunits, e.g., chimera a61e207a3208e474 has 1e207 amino acids of a6 and 208e474 of a3. C) Depicts mutations of a6 subunits made in the extracellular domain (Dash and Lukas, 2012), transmembrane domain a helices M1 (Ley et al., 2014; Jensen et al., 2013) and M2 (Dash and Lukas, 2012), and the intracellular domain (Henderson et al., 2014), as well as mutations of b2 subunits to increase ER exit (Henderson et al., 2014) and b3 subunits (Dash et al., 2014).

A strategy for expressing AChRs of defined subunit composition and order is to link subunits into concatamers (Zhou et al., 2003). This approach overcame the possible need for a specific chaperone in expressing a6b2b3* AChRs in oocytes, and permitted expression of complex (a6b2)(a4b2)b3 and (a4b2)(a6b2)b3 subtypes with defined subunit orders as well as (a6b2)2b3 (Fig. 3; Kuryatov and Lindstrom, 2011). Concatameric a6b2b3* AChRs exhibited the expected pharmacological properties, such as high sensitivity to nicotine and blockage by a-CtxMII (Table 2). In the case of (a6b2)(a4b2)b3 AChRs, pairing b3 subunit with either a6 or a4, did not significantly alter the pharmacology (Table 2). 4.2. Expression in mammalian cell lines Expression of a6* AChRs in transfected cell lines has proven especially challenging. In contrast with oocytes, even a6b4 is hard to express in HEK cell lines. Fucile et al. (1998) expressed chick a6b4

and a6b2 in modified HEK cells and achieved less than 25% of cells expressing functional AChRs for both subtypes. We found that b3 increased surface expression of human a6b4 and a6b2 in cells, but still not sufficiently to detect function (Tumkosit et al., 2006). Some of the strategies used in oocytes, such as chimeras and gain-of-function mutagenesis, have been successfully applied to expression of a6b2* and a6b4* AChRs in HEK cells (Table 3). Chimeras of a6 with the large cytoplasmic loop and transmembrane domains of a3 or a4 are functional when expressed in HEK cells to form (a6b4)2b3 AChRs (Evans et al., 2003). For functional expression of a6b4 and (a6b4)2b3 AChRs, such chimeric modification can be further reduced to replacement of a small C-terminal segment in the intracellular M3-M4 loop of a6 with that of a3 as illustrated in Fig. 2 (Jensen et al., 2013, 2014). The M211L mutation of a6, which is good for functional expression of (a6b2)2b3 AChRs in oocytes (Ley et al., 2014), is less efficient than F223L in functional expression of (a6b4)2b3 in cells (Jensen et al., 2013). Chimeras of a6, together

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J. Wang et al. / Neuropharmacology 96 (2015) 194e204

Fig. 3. Illustration of a6b2* AChRs made from concatamers. Pentameric (a6b2)2b3 was constructed by connecting the C terminus of one subunit to the N terminus of another subunit using AGS sequences repeated 6 or 12 times in the following order: b3, a6, b2, a6, b2, as shown on the left (Kuryatov and Lindstrom, 2011). Pentameric (a6b2)(a4b2)b3 and (a4b2)(a6b2)b3 AChRs that differ in b3 linkage to a6 or a4 were constructed similarly. These two AChR subtypes displayed similar pharmacological properties when expressed in oocytes and were similar to brain AChRs (Salminen et al., 2007; Kuryatov and Lindstrom, 2011).

with a gain-of-function b3 mutation, produced functional AChRs in HEK cells, sufficient for high-throughput drug development (Capelli et al., 2011). However, these modified (a6b2)2b3 AChRs with a hyperactive b3 subunit, displayed higher sensitivity to agonist, and slower desensitization rate compared to wild type AChRs (Rasmussen et al., 2014), thus are of limited value for explaining normal a6* AChR physiology or developing a6* drugs.

a6b2* AChRs were recently expressed in neuroblastoma 2a (N2a) cells with the help of a fused enhanced green fluorescent protein (eGFP) in the cytoplasmic loop of a6, as illustrated in Fig. 2 (Xiao et al., 2011; Henderson et al., 2014). This fluorescent protein facilitated detection of cells that expressed a6* AChRs. By deleting an endoplasmic reticulum (ER) retention sequence and engineering in another ER exporting sequence in b2 subunit, Lester's group was

Table 2 Physiological characterization of a6b2* AChRs in oocytes and cell lines. Subtype

Modification

Species

System

Drug

a6b 2

Wild type Wild type a61e207a3208e474

Human Chick Human

Oocyte HEK Oocyte

a61e207a4208e594

Human

Oocyte

a61e207a3208e474 eGFP in a6 eGFP in a6a

Human Mouse Mouse

Oocyte N-2a N-2a

ACh ACh ACh Nicotine ACh Nicotine Nicotine ACh ACh

Wild type b3ea6eb2ea6eb2

Human Human

Oocyte Oocyte

eGFP in a6d

Mouse

N-2a

a61e207a3208e474 a6M211L 1e297 a3298e437a6426e464

Human Human

HEK Oocyte

a61e305a3306e437a6426e464 a61e297a3298e474

Human Human

Oocyte Oocyte

b3 gain of function a61e207a3208e474, b3 GOF a61e207a3208e474, b3 GOF

Human Human Human

Oocyte HEK HEK

a61e207a3208e474, b3 GOF a6N143DþM145V GOF

Human Human

HEK Ooctye

a6a4b 2

Wild type

Human

a6a4b2b3

b3ea6eb2ea4eb2

Human

b3ea4eb2ea6eb2

Human

a6b2b3

EC50 (mM)

Average current or Emax

Reference

14 ± 1 4.2 ± 0.9 56 ± 9 3.2 ± 0.5 13

12 nAb 31 ± 6 pA About 1 mA 16% About 1 mA 10% 48 ± 12 nA 54 ± 10 pAc 70 ± 11 pAc

Broadbent et al., 2006 Fucile et al., 1998 Kuryatov et al., 2000

4 nAb

ACh ACh Nicotine ACh Nicotine ACh ACh Nicotine ACh ACh Nicotine ACh Nicotine ACh Nicotine ACh Nicotine

0.04 0.94 0.14 0.23 0.02

Ooctye

ACh Nicotine

2.6 ± 0.2 1.3 ± 0.1

Ooctye

ACh Nicotine ACh Nicotine

0.828 ± 0.047 0.170 ± 0.079 1.76 ± 0.25 0.397 ± 0.122

Ooctye

1.21 ± 0.09 0.387 ± 0.051 0.31 ± 0.10 0.12 ± 0.08 6.4 1.33 ± 0.25 0.203 ± 0.054 0.756 ± 0.262 0.711 ± 0.120

Kuryatov et al., 2000 Dash et al., 2011b Xiao et al., 2011 Xiao et al., 2011 Broadbent et al., 2006 Kuryatov and Lindstrom, 2011

24.4 ± 0.9% Henderson et al., 2014

Up to 1 mA 30.7 ± 26.7 150e250 nAb 27.5 ± 1.6 120 ± 17 nAb

Rasmussen et al., 2014 Ley et al., 2014 Jensen et al., 2013 Ley et al., 2014 Broadbent et al., 2006 Capelli et al., 2011a Rasmussen et al., 2014

65% 98 ± 21 nA

Rasmussen et al., 2014a Dash and Lukas, 2012 Kuryatov et al., 2000 Kuryatov and Lindstrom, 2011

78.8 ± 9.5% Kuryatov and Lindstrom, 2011 58.3 ± 7.3%

Data were obtained by electrophysiological assay except where noted as [a]. Emax: maximum efficacy relative to ACh. Amino acid sequence of chimera is numbered as corresponding subunits, i.e.: chimera a61e207a3208e474 has 1e207 amino acids of a6 and 208e474 of a3. [a] is acquired from FLIPR-type assay. Single ACh doses were applied in some cases, noted as [b] (1 mM) or [c] (300 mM ACh). [d]: a modified b2 for ER exporting was used for expression. GOF: gain-of-function mutation.

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Table 3 Physiological characterization of a6b4* AChRs in oocytes and cell lines. Subtype

Modification

Species

System

Drug

EC50 (mM)

a6b 4

Wild type

Mixc

Oocyte

Mixd Chick Human

Oocyte HEK Oocye

28 ± 4 22 ± 3 37 105 ± 8 18 ± 5 7.1 ± 2.6

Human Mouse

Oocyte Oocyte

a6F223L or a6M211L/F223L a61e207a4208e594 a61e207a4208e594

Human Human Human

HEK Oocyte HEK

a61e207a3208e474 a61e207a3208e474 a61e207a3208e474 a61e305a3306e437a6426e464 a6F223L 1e305a3306e437a6426e464, a61e400a3412e437a6426e464

Human Human Human Human Human Human

Oocyte HEK Oocyte HEK Oocyte HEK

ACh Nicotine ACh ACh ACh Nicotine ACh ACh Nicotine ACh ACh ACh Nicotine Nicotine ACh ACh ACh ACh ACh

Wild type

Human

Oocyte

b3 b3E221DþF223V b3 gain of function b3 GOF

Human Mouse Mouse

Oocyte Oocyte Oocyte

Human Human

Oocyte Oocyte

b3 GOF

Mouse

Oocyte

a6 gain of function a6 GOF

Mouse Human

Oocyte Oocyte

a6b4b3

S144NþS148V

ACh Nicotine ACh ACh ACh ACh ACh Nicotine ACh Nicotine Nicotine Nicotine

38 26 9.6 or 13 37 ± 12 11 ± 2 18 ± 4 25 14

Average current or Emax 18% 66 ± 13 pA 80 nA 25% 49 ± 15 nAb 65 ± 25 nA 27 ± 7 nA 0.7e18.1 mA 58 ± 4% 224 ± 28 nA 3e4 mAb

2.4 Up to 10 mAb 4.7 33 ± 8 10 ± 3 6.6 10 6.5 0.43 0.42 0.42 0.07 1.2 0.89

Reference Gerzanich et al., 1997

300 nA 20% 18 ± 5.6 nAb 85 ± 15 nA 266 ± 27 nA 307 ± 32 nA 560 ± 140 nAb 2.1 ± 0.2 mA 1.9 ± 0.3 mA 2.1 ± 0.2 mA 2.5 ± 0.3 mA 0.8 ± 0.2 mA 350 ± 52 nA

Gerzanich et al., 1997 Fucile et al., 1998 Kuryatov et al., 2000 Broadbent et al., 2006 Dash et al., 2011a Jensen et al., 2013a Evans et al., 2003 Evans et al., 2003 Dash et al., 2011b Jensen et al., 2013a Jensen et al., 2013 Jensen et al., 2013a Jensen et al., 2013 Jensen et al., 2013a Kuryatov et al., 2000 Broadbent et al., 2006 Dash et al., 2014 Dash et al., 2014 Broadbent et al., 2006 Dash et al., 2011a Dash et al., 2011a Dash and Lukas, 2012 Dash and Lukas, 2012

Data were obtained by electrophysiological assay except where noted as [a]. Emax: maximum efficacy relative to maximum responses of ACh. Amino acid sequence of chimera is numbered as corresponding subunits, i.e.: chimera a61e207a3208e474 has 1e207 amino acids of a6 and 208e474 of a3. [a] was acquired from FLIPR-type assay. Single ACh doses (1 mM) were applied in some cases, noted as [b]. A mixed species of DNA was used in some cases, noted as [c] (chick a6 and human b4) or [d] (rat a6 and human b4). GOF: gain-of-function mutation.

able to increase the percentage of cells with expression of functional a6b2 AChRs from 26% to 55% (Srinivasan et al., 2011; Xiao et al., 2011). b3 subunit increased expression of a6b2 AChRs generated from this fluorescent-labeled a6 expressed with modified b2 subunit with an ER transport signal. This led to whole cell and single channel functional characterization of (a6b2)2b3 AChRs in N-2a cells (Henderson et al., 2014). Kracun et al. (2008) showed that chimeras of various AChRs with a large cytoplasmic domain from other subunits can alter assembly, transport, and function. Substitution of the entire cytoplasmic domain of a6 with a3 slows the desensitization of a6b4 AChRs by ACh (Jensen et al., 2014). Thus, the cytoplasmic region is clearly important. Large modifications in it like inserting a large fluorescent protein might also alter assembly, transport, or function. 4.3. Chaperoning expression of cloned a6* AChRs As mentioned in Sections 2 and 3, a6* AChRs are minor brain subtypes with expression restricted mainly to mid-brain dopamine neurons and the visual system (Le Novere et al., 1996; Quik et al., 2000; Han et al., 2000; Whiteaker et al., 2000; Champtiaux et al., 2002). Such limited expression could be due to limited expression of chaperones required for a6* AChR assembly. Similarly, absence of chaperones could account for difficulties of expressing a6b2* AChRs in cultured cell lines. This is the case for other AChR subtypes. For example, expressing a7 AChRs in mammalian cells is extremely hard without the help of the chaperone RIC-3 (resistance to inhibitors of cholinesterase-3) (Castillo et al., 2005; Lansdell et al., 2005; Williams et al., 2005). Although many chaperones

have been identified for various AChR subtypes to facilitate subunit folding and assembly, enhance trafficking, or prevent proteasome degradation (as reviewed in Jones et al., 2010; Colombo et al., 2013), none of them has been reported positive for a6* AChRs. This may be partially due to lack of a good way to express recombinant a6 in vitro to fish out the right chaperones. The factors that retard surface expression of a6b2* AChRs are: 1) a6 and b2 subunits tend to form partially assembled intermediates; 2) a6 is vulnerable to degradation inside HEK cells; 3) b3 does not assemble efficiently with a6b2 AChRs (Tumkosit et al., 2006; Ley et al., 2014). Several chaperones have been identified to facilitate assembly and trafficking of AChRs, such as RIC-3, UBXD4, UNC-50, VILIP-1, 14-3-3 and etc. (Castillo et al., 2005; Lansdell et al., 2005; Williams et al., 2005; Rezvani et al., 2009; Eimer et al., 2007; Lin et al., 2002; Exley et al. 2006; Jeanclos et al., 2001). Together with modification of AChR subunits mentioned in Sections 4.1 and 4.2, these chaperones and others yet to be discovered could be useful to increase surface expression of a6b2* AChRs in mammalian cells and understand their neuropharmacological relevance in brain. Pharmacological chaperons such as nicotine increase the number of AChRs in brain and cultured cells (Marks et al., 2011; Henderson et al., 2014; Wüllner et al., 2008; McCallum et al., 2006a,b; Kuryatov et al., 2005; Sallette et al., 2005). Although upregulation of a6b2* AChR by nicotine is controversial in brain (see Section 3.2), we and others agree that nicotine increases a6* AChRs both inside and on the plasma membrane of mammalian cells in tissue culture (Tumkosit et al., 2006; Walsh et al., 2008; Henderson et al., 2014). Unfortunately, nicotine is of limited use for expressing complex a6a4* AChRs subtypes, because it is more

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potent for upregulating a4b2* rather than a6b2* AChRs (Kuryatov et al., 2005; Tumkosit et al., 2006; Walsh et al., 2008). But it could still be helpful for expressing a6(non a4)* AChRs. Overnight exposure to 100 mM nicotine increased both total and surface numbers of a6b2 and a6b2b3 AChRs by two to four fold in HEK cells (Tumkosit et al., 2006). In N-2a cells, 50 nM nicotine, a physiological relevant concentration in smokers (Matta et al., 2007), accelerated the insertion of a6b2b3 AChRs in the plasma membrane and increased surface expression by three fold (Henderson et al., 2014). These data suggest that nicotine facilitates assembly and trafficking of a6* AChRs in mammalian cells, which may help to account for the addictive effect of nicotine and lower risk for Parkinson's disease among smokers (Srinivasan et al., 2014). 5. Summary

a6b2b3* AChRs are neuropharmacologically important subtypes in the brain and pharmaceutical targets for smoking cessation and Parkinson's disease (Crooks et al., 2014; Srinivasan et al., 2014). It is challenging to express them in cell lines. Concatameric linkers fulfilled this goal in Xenopus oocytes (Kuryatov and Lindstrom, 2011), and were used in functional expression of GABAA receptors in mammalian cells (Akk et al., 2009). (a6b2)2b3 AChRs have been expressed from free subunits in oocytes after small modifications of the a6 subunit (Ley et al., 2014). Expression of (a6b2)2b3 AChRs in cell lines should be possible with variations on this theme. Expressing (a6b2)(a4b2)b3 AChRs in cell lines will require either variations like concatamers used in oocytes or the discovery of chaperones that promote efficient assembly of this complex subtype while suppressing expression of other subtypes that could be formed from the same mix of subunits. Acknowledgments We thank Dr. Jie Luo for useful comments on the manuscript. This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS11323] and the National Institutes of Health National Institute on Drug Abuse [Grant DA030929]. References Akk, G., Covey, D.F., Evers, A.S., Steinbach, J.H., Zorumski, C.F., Mennerick, S., 2009. The influence of the membrane on neurosteroid actions at GABA(A) receptors. Psychoneuroendocrinology (Suppl. 1), S59eS66. Azam, L., Winzer-Serhan, U.H., Chen, Y., Leslie, F.M., 2002. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. J. Comp. Neurol. 444, 260e274. Azam, L., Dowell, C., Watkins, M., Stitzel, J.A., Olivera, B.M., McIntosh, J.M., 2005. Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors. J. Biol. Chem. 280, 80e87. Azam, L., Maskos, U., Changeux, J.P., Dowell, C.D., Christensen, S., De Biasi, M., McIntosh, J.M., 2010. Alpha-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between a6b4 and a6b2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release. FASEB J. 24, 5113e5123. Baddick, C.G., Marks, M.J., 2011. An autoradiographic survey of mouse brain nicotinic acetylcholine receptors defined by null mutants. Biochem. Pharmacol. 82, 828e841. Bansal, A., Singer, J.H., Hwang, B.J., Xu, W., Beaudet, A., Feller, M.B., 2000. Mice lacking specific nicotinic acetylcholine receptor subunits exhibit dramatically altered spontaneous activity patterns and reveal a limited role for retinal waves in forming ON and OFF circuits in the inner retina. J. Neurosci. 20, 7672e7681. Beiranvand, F., Zlabinger, C., Orr-Urtreger, A., Ristl, R., Huck, S., Scholze, P., 2014. Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex. Br. J. Pharmacol. http://dx.doi.org/10.1111/bph.12841 (in press). Broadbent, S., Groot-Kormelink, P.J., Krashia, P.A., Harkness, P.C., Elgoyhen, N.S., Beato, M., Sivilotti, L.G., 2006. Incorporation of the b3 subunit has a dominantnegative effect on the function of recombinant central-type neuronal nicotinic receptors. Mol. Pharmacol. 70, 1350e1357. Brunzell, D.H., Boschen, K.E., Hendrick, E.S., Beardsley, P.M., McIntosh, J.M., 2010. aConotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus

accumbens shell regulate progressive ratio responding maintained by nicotine. Neuropsychopharmacology 35, 665e673. Brunzell, D.H., McIntosh, J.M., Papke, R.L., 2014. Diverse strategies targeting a7 homomeric and a6b2* heteromeric nicotinic acetylcholine receptors for smoking cessation. Ann. N. Y. Acad. Sci. http://dx.doi.org/10.1111/nyas.12421 (in press). Capelli, A.M., Castelletti, L., Chen, Y.H., Van der Keyl, H., Pucci, L., Oliosi, B., Salvagno, C., Bertani, B., Gotti, C., Powell, A., Mugnaini, M., 2011. Stable expression and functional characterisation of a human nicotinic acetylcholine receptor with a6b2 properties: discovery of selective antagonists. Br. J. Pharmacol. 163, 313e329. Cartier, G.E., Yoshikami, D., Gray, W.R., Luo, S., Olivera, B.M., McIntosh, J.M., 1996. A new a-conotoxin which targets a3b2 nicotinic acetylcholine receptors. J. Biol. Chem. 271, 7522e7528.
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n, F., Gerber, S., Sala, S., Castillo, M., Mulet, J., Gutie Sala, F., Criado, M., 2005. Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors. J. Biol. Chem. 280, 27062e27068. Champtiaux, N., Han, Z.Y., Bessis, A., Rossi, F.M., Zoli, M., Marubio, L., McIntosh, J.M., Changeux, J.P., 2002. Distribution and pharmacology of a6-containing nicotinic acetylcholine receptors analyzed with mutant mice. J. Neurosci. 22, 1208e1217. Champtiaux, N., Gotti, C., Cordero-Erausquin, M., David, D.J., Przybylski, C., Lena, C., Clementi, F., Moretti, M., Rossi, F.M., Le Novere, N., McIntosh, J.M., Gardier, A.M., Changeux, J.P., 2003. Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. J. Neurosci. 23, 7820e7829. Champtiaux, N., Changeux, J.P., 2004. Knockout and knockin mice to investigate the role of nicotinic receptors in the central nervous system. Prog. Brain Res. 145, 235e251. 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