Neuropharmacology 96 (2015) 194e204
Contents lists available at ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
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.
J. Wang et al. / Neuropharmacology 96 (2015) 194e204
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.
195
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.
196
J. Wang et al. / Neuropharmacology 96 (2015) 194e204
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; Hernandez-Vivanco et al., 2014). a3a6* Hone et al., 2012; Pe AChRs are a small population of a6* AChRs (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
197
of other AChR subtypes in visual nuclei (Champtiaux et al., 2002; Gotti et al., 2005b), but a small (
Contents lists available at ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
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.
J. Wang et al. / Neuropharmacology 96 (2015) 194e204
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.
195
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; Hernandez-Vivanco et al., 2014). a3a6* Hone et al., 2012; Pe AChRs are a small population of a6* AChRs (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 (
