B RA I N R E SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s r e v

Review

Regulation of neuronal nicotinic receptor traffic and expression

Annalisa Gaimarri a,1 , Milena Moretti a,1 , Loredana Riganti a,1 , Alessio Zanardi b , Francesco Clementi a,, Cecilia Gotti a
a CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy b Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, Modena, Italy

A R T I C LE I N FO Article history: Accepted 13 February 2007 Available online 24 February 2007 Keywords: Neuronal nicotinic receptor Subtypes Nicotine Up-regulation 1,2-bis-N-cytisinylethane (CC4)

AB S T R A C T Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of cation channels widely distributed in the brain, whose subunit composition and biophysical properties vary depending on the subtype and the area of the brain in which they are found. Brain nAChRs are also the target of nicotine, the most widespread drug of abuse. Chronic nicotine exposure differentially affects the number, subunit composition, stoichiometry and functional state of some nAChR subtypes, leaving others substantially unaffected. In this review, we will summarise recent data concerning the nAChR subtypes expressed in the CNS, and how they are regulated by means of chronic nicotine and/or nicotinic drugs. We will particularly focus on the possible mechanisms involved in the up-regulation of nAChRs. 2007 Elsevier B.V. All rights reserved.

Contents
1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brain nicotinic receptor subtypes . . . . . . . . . . . . . . . . . 2.1. Structure of nAChRs. . . . . . . . . . . . . . . . . . . . . 2.2. Subunit composition and localisation of nAChR subtypes 3. Nicotine regulation of nicotinic receptor subtypes . . . . . . . . 3.1. In vitro studies. . . . . . . . . . . . . . . . . . . . . . . . 3.2. In vivo studies . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Possible mechanisms involved in nAChR up-regulation . 4. Up-regulation of nAChRs by a novel nicotinic antagonist . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 135 135 136 137 137 138 138 140 141 141

Corresponding author. Fax: +39 02 7490574. E-mail address: Francesco.clementi@unimi.it (F. Clementi). 1 The three authors contributed equally to this work. 0165-0173/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2007.02.005

B RA I N RE SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

135

1.

Introduction

This review will summarise recent data concerning nAChR subtypes expressed in the CNS, and how they are regulated by chronic nicotine administration and nicotinic drug treatment.

Neurones communicate with each other at highly specialised contact sites called synapses which consist of a presynaptic nerve terminal and a post-synaptic neuron. Synaptic transmission is fundamental to many brain processes and its strength can be enhanced or diminished by cell activity. This plasticity depends on mechanisms that regulate the strength of synaptic transmission at the levels, inter alia, of neurotransmitter release from synaptic terminals and the concentration of neurotransmitter receptors in post-synaptic neurones. Different lines of evidence indicate that both plasma membrane domains and matrix-bound receptor-associated proteins are crucial for the localisation and anchoring of receptors in the pre- and post-synaptic membrane of central synapses. It has been found that various receptors and enzymes associate with cytosolic and cytoskeletal proteins, via PDZ-domain proteinprotein interactions, which not only allow the recruitment of ionotropic and metabotropic receptors at the synapse, but also the assembly of signalling cascades and enzyme complexes at synaptic sites. Receptor localisation and trafficking in neurones are thus fundamental mechanisms involved in synaptic plasticity, the pathophysiology of diseases and drug activities. Receptors trafficking refers to their translocation from the endoplasmic reticulum (ER) to a plasma membrane domain, to their internalisation from plasma membrane to different cytoplasmic compartments and finally the lateral movements that regulate and control receptor aggregation at synaptic and extrasynaptic sites (Green and Millar, 1995; Kittler and Moss, 2001). Receptor traffic is particularly complex in the case of ligand-gated ion channels, consisting of multimeric proteins formed by different subunits, because very strict quality control is required in the early steps of subunit assembly with appropriate subunit pairing in order to ensure that functional receptors can reach the surface membrane (Green and Millar, 1995). In the nervous system, the neurotransmitter acetylcholine (ACh) binds to metabotropic muscarinic and ionotropic nicotinic receptors (nAChRs), interactions that are recognised as being highly relevant to several functions including cognition, locomotion and analgesia (Champtiaux and Changeux, 2002; Drago et al., 2003; Picciotto et al., 2000, 2001). In particular, the interactions of ACh with nAChRs have recently been reevaluated. nAChRs in the CNS are mainly located presynaptically and modulate the release of almost all neurotransmitters, but they have a post-synaptic localisation in some areas, where they mediate fast synaptic transmission (Dajas-Bailador and Wonnacott, 2004; Gotti and Clementi, 2004; Jensen et al., 2005). nAChRs are also the targets of nicotine (the most common drug of abuse) whose complex activities in the nervous system are due to its ability to mimic the activity of ACh on this receptor subset. The different effects of nicotine are determined by the functional features and location of the nAChR subtypes with which it interacts in specific neuronal systems (Gentry and Lukas, 2002; Laviolette and van der Kooy, 2004).

2.
2.1.

Brain nicotinic receptor subtypes

Structure of nAChRs

Ionotropic neuronal nAChRs are a very heterogeneous class of receptor subtypes consisting of the combinations of different subunits encoded by distinct genes. The genes that have been cloned so far are divided into two subfamilies of nine (210) and three (24) subunits and are expressed in the nervous system, cochlea and a number of non-neuronal tissues (Gotti and Clementi, 2004; Hogg et al., 2003). The topology of the receptor subunits consists of a large extracellular N-terminus domain, four transmembrane domains and an intracellular loop. nAChRs are a very heterogeneous family of subtypes formed by the pentameric assembly of identical (homomeric receptors) or homologous subunits (heteromeric receptors) around a central ion pore and have different structural functional and pharmacological properties (Le Novere et al., 2002; Lindstrom, 2000). Two main classes have been identified: the Bungarotoxin (Bgtx)-sensitive receptors, which are made up of the 7, 8, 9 and/or 10 subunits and can form homomeric or heteromeric receptors, and Bgtx-insensitive receptors, which are heteromeric receptors that consist of 26 and 24 subunits, and bind nicotine and many other nicotinic agonists with high affinity but not Bgtx (Gotti and Clementi, 2004; Lindstrom, 2000). The ACh binding site lies at the interface between an and an adjacent subunit which may be the same subunit in homomeric receptors (7, 8), a different subunit in heteromeric Bgtx-sensitive receptors (78 or 910) or a subunit in heteromeric Bgtx-insensitive receptors (Gotti et al., 2006). The ACh binding site has a primary and a complementary component. In heteromeric Bgtx-insensitive nAChRs, the primary component is carried by the 2, 3, 4 or 6 subunits and the complementary component by the 2 or 4 subunits whereas, in homomeric 7 receptors, each subunit contributes to both the primary and complementary components of the binding site (reviewed in Changeux and Edelstein, 1998; Corringer et al., 2000). The 5 and 3 subunits carry neither the principal nor the complementary component of the ACh binding site, but form functional channels in heterologous systems only when they are expressed with another ligand binding subunit and a complementary subunit; it is generally assumed that they do not participate directly in the formation of the ligand binding site, but may occupy a position that is comparable with that of the muscle 1 subunit in assembled receptors (Broadbent et al., 2006; Le Novere et al., 2002). These subunits may contribute to the receptor targeting and localisation in neuronal plasma membrane domains (Gotti et al., 2005; Le Novere et al., 2002). The recent crystallisation of the ACh binding protein from Lymnaea stagnalis snails (a homopentameric soluble protein with an affinity spectrum resembling that of homomeric 7 or

136

B RA I N R E SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

9 receptors) bound with nicotine or carbamylcholine and the ACh binding protein from the Aplysia salt water mollusc bound with lobeline or epibatidine has greatly helped to define nAChR binding sites (Celie et al., 2004; Hansen et al., 2005; Sine and Engel, 2006) and revealed the basis for agonist recognition site in nAChRs. In addition to their classical ACh binding sites, nAChRs have a number of allosteric binding sites. Various structurally diverse ligands can bind to these sites and modify nAChR function (reviewed in Jensen et al., 2005). Electrophysiological and biochemical studies of heterologously expressed 42 subtypes have shown that they have a pentameric structure but, difference in the 4:2 subunit ratio can lead to subtypes with different pharmacological and functional properties. In particular, it is found in two different stoichiometries: the (4)3(2)2 subtype with low affinity for ACh (an EC50 of approximately 100 M), and the (4)2(2)3 subtype, with high affinity (an EC50 of approximately 1 M) (Moroni et al., 2006). The (4)3(2)2 stoichiometry predominates in transfected mammalian cells, but long-term exposure to nicotine, boosting the number of 2 subunits by transfection, or culturing the cells at 29 C can all promote the expression of the (4)2(2)3 subtype with high ACh sensitivity (Nelson et al., 2003). Functional studies of thalamic 42 receptors suggest that both stoichiometries may be present in vivo, but no biochemical proof of this has yet been obtained (Marks et al., 1999). In addition of being permeable to monovalent Na+ and K+ ions, the Bgtx-sensitive and insensitive receptors are both permeable to Ca2+ and their permeability is influenced by their subunit composition (reviewed in Fucile, 2004). In neurones, Ca2+ influx after nAChR stimulation takes place

by means of two mechanisms: Ca2+ influx through the nAChRs, and Ca2+ entry through voltage-activated Ca2+ channels activated by plasma membrane depolarisation due to Na+ influx through nAChRs. These two mechanisms can be physiologically complementary and play important roles in cell signalling by activating different downstream intracellular pathways in neurones (reviewed in Dajas-Bailador and Wonnacott, 2004).

2.2. Subunit composition and localisation of nAChR subtypes

The identification of the different native nAChR subtypes and their specific roles was initially hampered by the lack of subtype-specific ligands and by the fact that many neuronal cells express multiple subtypes but the recent availability of genetically engineered knockout (Ko) or knockin (Kin) mice has made it possible to analyse the pharmacology and functional role of nAChRs in complex neurobiological systems and correlate major phenotypes with individual nAChR subtypes. In defining heteromeric subtypes, an asterisk indicates that unidentified nAChR subunits may be present in the receptor complex; when all five subunits are identified, the subtype has been exactly defined (see also Fig. 1). nAChRs are widely distributed in the brain, and their subunit composition is cell- and region-specific. The large majority of nAChRs in the nervous system contain one type of and one type of subunit, with 42 receptors accounting for 90% of the high affinity neuronal nAChRs in mammalian brain, and 34 receptors being the major subtype in the autonomic ganglia and adrenal medulla as well as in subsets of CNS neurones in the medial habenula, dorsal medulla,

Fig. 1 Summary of the data on nAChR subtype localisation in rodent CNS. The data shown bring together results of binding, immunoprecipitation and/or immunopurification experiments in the cortex, cerebellum, hippocampus, interpeduncular nucleus, medial habenula and pineal gland, and the results of in situ hybridisation, single-cell PCR or binding studies of the amygdala, hypothalamus, locus coeruleus, olfactory bulb, raphe nuclei, spinal cord, substantia nigraventral tegmental area and thalamus obtained from rat and/or WT and/or nAChR subunit Ko mice (Champtiaux et al., 2003; Gotti et al., 2005; Hogg et al., 2003).

B RA I N RE SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

137

pineal gland and retina (Gotti and Clementi, 2004) (Fig. 1). Both the 42 and 34 subtypes may also contain the 5 subunit, whose presence is believed to increase the rate of channel desensitisation and calcium permeability (Gotti and Clementi, 2004; Gentry and Lukas, 2002; Lindstrom, 2000). In addition to these major central and peripheral nAChR subtypes, many other native nAChR subtypes with more complex subunit compositions have been recently identified in the CNS. In agreement with the findings of in situ hybridisation experiments showing the selective localisation of 6 mRNA in the substantia nigra and ventral tegmental area, the locus coeruleus and the retina (Fig. 1), immunopurification and immunoprecipitation studies have identified the 6 receptors (6423 and 623 subtypes) at molecular level in rodent striatum, retina, the superior colliculus and nucleus genicolatus lateralis. The 6 receptors in the mesostriatal and visual pathways have pre-synaptic localisation as it has been shown that lesions induced by 6-hydroxydopamine and eye enucleation lead to their disappearance from the striatum, and the target retina tissues (the superior colliculus and nucleus genicolatus lateralis) (Champtiaux et al., 2003; Moretti et al., 2004). The expression of 2 mRNA is limited in rodent brain, with a moderate signal being observed in the retina and interpeduncular nucleus (IPN) (Drago et al., 2003). Immunopurification experiments have confirmed the presence of 262, 242 and 22 subtypes in retina (Moretti et al., 2004) and an 22 subtype in the IPN (Gotti and Zoli unpublished results). In agreement with the mRNA data, cortical 2 subtype expression is species-specific amongst mammals: being almost undetectable in rodents but present in 21% of the high affinity epibatidine receptors in monkey, where it forms an 242 subtype (Quik et al., 2005); it is also present in 10% of the receptors in Brodmann area 21 of human cortex (Gotti et al., 2006). The use of the selective antagonist Bgtx has allowed the localisation of 7-containing receptors which are highly expressed in the cortex, hippocampus and subcortical limbic regions of the brain (see Fig. 1), and at low levels in the thalamic regions and basal ganglia. It has been reported that the 7 subunits co-assemble with the subunits of the Bgtx-insensitive subfamily (2 or 3) in heterologous systems, but no biochemical evidence of such an assembly has yet been obtained in neurones (Khiroug et al., 2002; Palma et al., 1999). The 8 subunit is present in the avian CNS, where it forms both homomeric 8 and heteromeric 78 receptors, but neither have been found in mammalian brain. The 9- and 10-containing receptors have widespread neuronal and non-neuronal localisations and have been found in the cochlea, dorsal root ganglia, the pars tuberalis of the pituitary gland, lymphocytes and keratinocytes (Gotti and Clementi, 2004). These receptors have recently been identified as possible targets for pain control (Vincler et al., 2006). 6 nAChRs are expressed at higher concentration on nerve terminals than in the cell body/dendrite compartment thus suggesting that the 6 subunit, or another subunit regularly associated with 6 (such as 3), plays a targeting

role. Support for a targeting role of 3 subunits comes from studies of 3 Ko mice showing that 6 nAChR expression is significantly reduced in both the dopamine cell body (ventral midbrain) (Gotti et al., 2005) and terminal regions (striatum), but more markedly in the latter (76% vs 42% in the ventral midbrain). This targeting role may be accomplished by the 5 subunit in some 4 receptors in the striatum (Champtiaux and Changeux, 2002). The physiological functions of nAChRs critically depend on the subtypes and their locations in the plasma membrane domains: i.e. the pre- or post-synaptic domain, the active zone or the extrasynaptic zone in the synapse. Little is known about the subcellular distribution of nAChRs in a single neuron, but the intracellular loop between transmembrane domains M3 and M4 of the nAChR subunits is critical for their localisation and/or targeting. Williams et al. (1998) have shown that the intracellular loop in the 3 subunit is fundamental for the synaptic targeting of 3-containing receptors in chick ciliary ganglion neuron, and Xu et al. (2006) have recently shown that the residue motifs in the intracellular loop are essential for the preferential targeting of the 7 subtype to the somatodendritic compartments in hippocampal culture, as well as for the axonal and dendritic domain localisation of the 42 subtype.

3. Nicotine regulation of nicotinic receptor subtypes

One generally accepted paradigm in cell membrane receptor studies is that the over-stimulation induced by agonists leads to a reduction in the number of cell surface receptors, whereas the prolonged inactivation induced by antagonists has the opposite effect (Gentry and Lukas, 2002). However, studies of the brains of tobacco smokers and animals chronically exposed to nicotine have shown that nAChRs apparently escape from these general rules because long-term exposure to nicotine often triggers an increase in the number of nAChRs (called up-regulation) (Gentry and Lukas, 2002; Perry et al., 1999). This particular effect of nicotine may be due to the fact that nicotine not only acts as an agonist but also as a timeaveraged antagonist, as demonstrated by functional studies of native and transfected nAChRs in which the constant presence of an agonist concentration leads to desensitisation: i.e. a progressive reduction in the nAChR response (Picciotto, 2003). The molecular mechanisms underlying the increased expression of cell surface nAChRs have been investigated in vitro and in vivo.

3.1.

In vitro studies

Cells transfected with nAChR subtypes show the nicotineinduced up-regulation of homomeric 7 and various heteromeric 32, 42, 62 and 34 receptors. Furthermore it has recently been shown that the presence of 3 subunits in 623 and 643 subtypes promotes greater nicotineinduced up-regulation than that observed in the 62 and 64 subtypes (Broadbent et al., 2006). This is not due to an increased affinity for nicotine, but probably to a direct effect of the 3 subunit on subtype assembly.

138

B RA I N R E SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

Studies of neuroblastoma cell lines expressing both 7 and 3 receptors have shown that they are both up-regulated by chronic nicotine treatment, but to a different extent and at higher doses than those necessary to up-regulate the 42 subtype, thus suggesting that the sensitivity and extent of the up-regulation of individual receptor subtypes can vary. In addition to nAChR subtype, up-regulation may depend on the host cell. For instance, studies of 42 and 7 transfected oocytes (Peng et al., 1997) have shown that nicotine exposure decreases their functional response; but studies of the 42 subtype that is stably expressed in HEK293 cells (Buisson and Bertrand, 2001) have found that chronic nicotine exposure does not desensitise the receptor. It is therefore possible that different host systems and/or their different protein repertoires can influence both the number and functions of the expressed subtypes.

types that may be differently sensitive to up-regulation because both autoradiographic and functional studies have shown that nicotine is incapable of up-regulating the number and function of 34 receptors in the IPN, medial habenula and other brain areas (Nguyen et al., 2004; Marks et al., 2004). In conclusion, all these studies indicate that, at molecular level, chronic nicotine exposure differentially affects the number (up-regulation), subunit composition, stoichiometry and functional status (desensitisation and inactivation) of some nAChR subtypes, leaving others substantially unaffected.

3.3. Possible mechanisms involved in nAChR up-regulation

Several models have been proposed to explain the different effects of nicotine on nAChR subtypes, but the mechanisms and cellular machinery required for nAChR up-regulation are still not completely understood. Before discussing the molecular mechanisms of receptor up-regulation, it is important to understand its cellular fate and traffic. Most of our knowledge of the up-regulation of nAChRs is based on studies on the 42 subtype which, in transfected cells and neurones, is localised on the cell surface and in intracellular pools, where it co-localises with a number of markers of the endoplasmic reticulum (ER). Each subunit is synthesised by membrane-bound polysomes and glycosylated with high-mannose; the subunits in the ER assemble into heteropentameric receptors with a high affinity for nicotine, and into a heterogeneous population of higher molecular weight species. Only the correctly assembled pentameric receptors exit from the ER to the Golgi, presumably after the masking of specific ER retention signals and the recognition of export motifs in properly folded and oligomerised 42 subunits (Ren et al., 2005). In the Golgi apparatus, the sugars are trimmed and processed into complex carbohydrates before reaching the cell surface. The surface receptors are internalised to the endosomal compartment and eventually targeted to the lysosomal compartment, where the receptors are degraded. Receptors assembly in the ER and their transport to the cell surface is a process of crucial importance in regulating the efficacy of synaptic transmission. It is a relatively inefficient process probably because of the tight quality controls that guarantee the functional competence of the final product: the cell surface receptors that are functionally required for membrane excitability in vivo. ER quality control operates at different levels and uses a variety of mechanisms. A central role is played by chaperone proteins that promote correct protein folding and assembly and prevent aggregation or the incorrect interaction of misfolded proteins with other proteins. A number of models have been proposed to explain the 42 subtype up-regulation of the observed in vivo and in vitro after chronic nicotine treatment: (i) an increase in receptor transport through the secretory pathway to the plasma membrane (Harkness and Millar, 2002)

3.2.

In vivo studies

Chronic nicotine treatment does not increase 2, 3, 4, 5 or 2 mRNA levels in mouse brain (Marks et al., 1992), which clearly suggests that post-transcriptional mechanisms are responsible for this effect. Studies of the brains of animals chronically treated with nicotine and those of human smokers (Gentry and Lukas, 2002; Perry et al., 1999; Tumkosit et al., 2006) have shown that the most up-regulated receptor is the 42 subtype. However, recent rodent studies have shown an increase in the number of 6 receptors in a rat model of nicotine self-administration (Parker et al., 2004) and a decrease in the number of striatal 6 receptors in mice and rats chronically treated with nicotine by means of minipumps (Lai et al., 2005; Mugnaini et al., 2006). These apparently discrepant results raise some concerns about the possible over-simple grouping of the results obtained in different species or using different protocols of nicotine administration. It is possible that receptor up-regulation depends on the concentration of nicotine and the duration of receptor exposure to it and that the different methods of nicotine administration lead to different brain nicotine levels and/or kinetics that can differently affect nAChRs. The data concerning nAChRs after chronic exposure to nicotine in vivo are also contradictory insofar as different functional assays have determined increased or decreased activity (Grilli et al., 2005). It has been reported that midbrain dopaminergic neurones are less responsive to nicotine (Pidoplichko et al., 1997) and that there is a decrease in dopamine release from striatal synaptosomes and 86Rb+ efflux from midbrain synaptosomes (Marks et al., 1993), whereas chronic anatoxin A (a nicotinic agonist) increases nicotine-stimulated dopamine release from rat striatum (Rowell and Wonnacott, 1990), and chronic nicotine administration in rats increases dopamine release from the striatum, decreases the release of ACh from the same area and the hippocampus, but has no effect on noradrenaline release (Yu and Wecker, 1994). Moreover, studies of 86Rb+ efflux from synaptosomes obtained from different brain areas of chronically exposed animals show that the increase in receptor number correlates well with the increase in functional activity (Nguyen et al., 2004). A further level of complexity in studying the in vivo effect of nicotine is due to the presence of different receptor sub-

B RA I N RE SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

139

does not require current flow through surface nAChRs because it occurs in the presence of the channel blocker mecamylamine and in the case of an 4 mutant that prevents nearly all nAChR functions. Another critical factor for receptor assembly and traffic control is subunit availability. Ficklin et al. (2005) have described the important role of ubiquilin-1 (an ubiquitin like protein that can interact with both the proteasome and ubiquitin ligase) in regulating nicotine-induced up-regulation: its binding to the 3 or 4 subunits sequesters them on proteasomes, thus decreasing the availability of subunits for the assembly/trafficking of mature receptors.

Fig. 2 Chemical structure of the cytisine extracted from the seeds of Laburnum anagyroides and 1,2-bis-N-cytisinylethane (CC4). (ii) a decrease in surface receptor internalisation due to nicotine-induced conformational modifications that prevent the removal of receptors from the cell surface (Peng et al., 1997) (iii) the isomerisation of surface receptors into a more easily activated high affinity conformation (Buisson and Bertrand, 2001; Vallejo et al., 2005) (iv) an increase in subunit assembly and/or a decrease in receptor turnover (Darsow et al., 2005; Kuryatov et al., 2005; Nashmi et al., 2003; Wang et al., 1998). Several lines of evidence suggest that a key step in nicotine-induced up-regulation is the assembly and trafficking of receptor subtypes and their expression on the cell surface. Recent studies by Sallette et al. (2004) have identified two segments in the extracellular domain of the 2 subunit (amino acids 7489 and 106116) that are critical for the upregulation of 2 subtypes. The amino acids in these segments form a compact microdomain that contributes to the subunit interface and faces the ACh binding site, and the authors suggest that nicotine binding to immature oligomers leads to a conformational reorganisation of the microdomain, strengthens the interactions between adjacent subunits, and facilitates the maturation process toward high affinity receptors (Sallette et al., 2004, 2005). This is in line with the findings of Kuryatov et al. (2005), who showed that nicotine acts as a pharmacological chaperone and that nAChR up-regulation

Fig. 3 (A) Drawing of the subcellular localisation of the high-affinity epibatidine-binding receptors present in control SH-SY5Y cells (C), and SH-SY5Y cells chronically treated with CC4 or nicotine (nic). Receptors are found on the cell membrane and in intracellular pools. The surface receptors were biotinylated followed by immunoprecipitation with streptavidin beads and then underwent Western blotting with anti-nAChR subunit-specific antibodies. (B) Western blotting of membrane and surface biotinylated receptors with anti-2 and anti-3 subunit-specific antibodies. There were no differences in the level of the 3 and 2 subunits present in total membranes obtained from control, CC4- or nicotine-treated cells, but a marked increase in the number of receptors containing the 3 and 2 subunits on the surface of CC4- and nicotine-treated cells.

140

B RA I N R E SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

Using two hybrid screening, Anand et al. have demonstrated that 4 subunits interact with the chaperone cytoplasmic protein 14-3-3 (Jeanclos et al., 2001) and the calcium sensor protein VILIP-1 (Lin et al., 2002) and that these interactions significantly increase the steady-state levels of the 4 subunit. This suggests that the greater availability of 4 subunit increases the formation of the 42 subtype, thus leading to an increase in surface 42 receptors. It has recently been shown that the newly cloned RIC-3 protein interacts with a number of nAChR subtypes and enhances their functional response depending on the subtype or the host system (Halevi et al., 2003; Lansdell et al., 2005; Williams et al., 2005). In conclusion, nicotine acts as a chaperone at subunit level and, by interacting or regulating interactions with chaperone proteins, can regulate the number and function of the 42 subtype.

4. Up-regulation of nAChRs by a novel nicotinic antagonist

Most of our knowledge of nAChR up-regulation comes from studies of the effects of nicotine on the 42 subtype, whereas much less is known about the effect of nicotine and nicotinic drugs on other subtypes. We have recently compared the effects of chronic treatment with nicotine or the novel cholinergic drug 1,2-bis-N-cytisinylethane (CC4) in SH-SY5Y neuroblastoma cells (which natively express both 3 and 7 AChRs and resemble human sympathetic neurones); and the controls were primary cultures of hippocampal neurones (which natively express the 42 and 7 subtypes) because both native cell types may conserve some endogenous regulatory mechanisms controlling receptor expression that are missing from transfected heterologous cells. CC4 is a nicotinic antagonist that belongs to a new class of compounds derived from cytisine (a potent full agonist of 34 and 7 receptors but only a partial agonist of 2 receptors)

(Chavez-Noriega et al., 1997), whose liposolubility has been increased by the introduction of aliphatic, alicyclic, arylalkylic or heteroarylic groups in the amino group (Carbonnelle et al., 2003). CC4 is a cytisine dimer (see Fig. 2) in which two cytisine moieties are connected by a polymethylene chain in such a way as to mimic nicotinic ligands (Boido et al., 2003; Canu Boido and Sparatore, 1999; Paton and Zaimis, 1949; Villarroya et al., 1996). Functional studies using the FLIPR technique (which exploits the Ca2+ transporting activity of nAChRs) (Carbonnelle et al., 2003), or electrophysiological recordings from patch clamp experiments using native nAChRs on SH-SY5Y cells, have shown that CC4 has lost the agonist activity of cytisine and becomes an antagonist of the 42 and 7 receptors expressed on transfected cells (Carbonnelle et al., 2003) and of the 3 receptors expressed on SH-SY5Y cells (Riganti et al., 2005). Chronic treatment with 100 M CC4 up-regulates the 42 subtype in hippocampal neurones, and the level of upregulation (2.2-fold) is very similar to that obtained using 100 M nicotine (2.5-fold). On the contrary, and in accordance with its lower affinity, a much higher concentration (1 mM) is necessary to up-regulate the 3 receptors on SH-SY5Y cells, the level of which was higher than that obtained in hippocampal neurones (3-fold vs 2.2-fold), and very similar to that obtained with chronic nicotine treatment (3.5-fold). Up-regulating the 7 receptors on both the hippocampal and SH-SY5Y cells requires the same high concentration of CC4 (1 mM) to give rise to a very similar (1.3-fold) increase in receptor number. Immunoprecipitation and immunopurification experiments using controls, and CC4- and nicotine-treated SHSY5Y cells, showed that the chronic treatments greatly increased the expression of subtypes containing the 2 subunit (32 and 324). As SH-SY5Y cells express heteromeric nAChRs on the cell membranes and in cytoplasm (Peng et al., 1997) we used the impermeant biotinylation reagent (sulpho-NHS-

Fig. 4 Possible mechanism for the up-regulation of 32 and 324 subtypes in SH-SY5Y neuroblastoma cells after chronic exposure to 1 mM CC4. In this model, we propose that CC4 permeates the cell membrane and reaches the ER, where it binds to immature oligomers containing the 2 subunit and elicits a conformational reorganisation of the binding microdomain that strengthens the interaction between adjacent 3 and/or 4 subunits, thus facilitating the maturation process toward the high affinity receptors that are transported to the Golgi and plasma membrane.

B RA I N RE SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

141

biotin) followed by immunoprecipitation with streptavidin beads to localise and quantify the up-regulated subtypes on the cell surface. As shown in Fig. 3, chronic treatment with nicotine or CC4 did not increase the levels of total 3 or 2 protein in the cell membranes, but greatly increased the surface expression of the receptors containing these subunits. These results, together with quantitative Northern blot data showing no change in the level of 3, 2 and 4 subunit mRNA in SH-SY5Y cells chronically treated with nicotine or CC4 (Riganti et al., 2005), strongly suggest that CC4 acts on posttranslational processes. Electrophysiological recordings in the presence of the subtype-specific antagonist -conotoxin MII confirmed that CC4 treatment selectively increases the 32-containing subtype on the cell surface, thus indicating that these upregulated receptors are functional (Riganti et al., 2005). Chronic CC4 treatment mainly up-regulates 2-containing receptors (the 32 and 324 subtypes) but not the 34 subtype, which is the most abundant subtype in SH-SY5Y cells (Sallette et al., 2004). These results are in agreement with previous data showing that nicotine up-regulates 32 to a much greater extent than 34 receptors, mainly because it promotes their maturation even in the absence of protein synthesis (Wang et al., 1998). As discussed above, Sallette et al. (2004, 2005) have recently identified the molecular mechanisms leading to the up-regulation of 2 subtypes and, by analogy, we can speculate that nicotine (or CC4) conformationally reorganises the binding microdomain that strengthens the interaction between adjacent subunits by binding to immature oligomers containing the 2 subunit and that this facilitates the maturation process toward high affinity receptors. As we and others (reviewed in Hogg et al., 2003) have found that both agonists and antagonists can induce receptor up-regulation, this process does not seem to be related to the same conformational change as that which occurs in channel opening. Given that one common characteristic of up-regulating drugs is cell permeability, we speculate that they may act after compartmentalisation within the intracellular milieu and that their binding (with specific pharmacological characteristics) to immature receptors can trigger a conformation favouring the maturation of 2 receptors. Fig. 4 shows our model in which the number of receptors on the cell surface and in the intracellular compartments after chronic exposure to CC4 is due to the more efficient assembly of 2 receptors allowing more of them to be transported to the cell surface. The fact that nicotinic drugs not only control nAChR channel opening, but also act on the pathway of receptor biosynthesis thus leading to an increase in the number of nAChRs on the cell surface (and possibly an increase in selected receptor subtypes), may change the response of a specific cell type to neurotransmitters and drugs. This may play an important role in synaptic plasticity and may therefore have therapeutic implications for diseases characterised by a reduction in the number of nAChRs. It may be possible in the future to design therapeutic interventions based on nicotinic ligands which, by increasing subunit assembly and/or rescuing misfolded or trafficking-defective nAChRs, can treat some neurodegenerative diseases such as Alzheimer's and Parkinson's disease whose pathogenesis is

also due to the aggregation and accumulation of misfolded proteins.

Acknowledgments
We thank Prof Michele Zoli for critically reading of the manuscript. This work was supported by the Italian PRIN 2005054943 (F. Clementi), Fondazione Cariplo grant No. 2004/1419 to F. Clementi; and FIRB (RBNE01RHZM) 2003 grant to C. Gotti.

REFERENCES

Boido, C.C., Tasso, B., Boido, V., Sparatore, F., 2003. Cytisine derivatives as ligands for neuronal nicotine receptors and with various pharmacological activities. Farmaco 58, 265277. Broadbent, S., Groot-Kormelink, P.J., Krashia, P.A., Harkness, P.C., Millar, N.S., Beato, M., Sivilotti, L.G., 2006. Incorporation of the beta3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors. Mol. Pharmacol. 70, 13501357. Buisson, B., Bertrand, D., 2001. Chronic exposure to nicotine upregulates the human (alpha)4(beta)2 nicotinic acetylcholine receptor function. J. Neurosci. 21, 18191829. Canu Boido, C., Sparatore, F., 1999. Synthesis and preliminary pharmacological evaluation of some cytisine derivatives. Farmaco 54, 438451. Carbonnelle, E., Sparatore, F., Canu-Boido, C., Salvagno, C., Baldani-Guerra, B., Terstappen, G., Zwart, R., Vijverberg, H., Clementi, F., Gotti, C., 2003. Nitrogen substitution modifies the activity of cytisine on neuronal nicotinic receptor subtypes. Eur. J. Pharmacol. 471, 8596. Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41, 907914. Champtiaux, N., Changeux, J.P., 2002. Knock-out and knock-in mice to investigate the role of nicotinic receptors in the central nervous system. Curr. Drug Targets. CNS Neurol. Disord. 1, 319330. 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, 78207829. Changeux, J.P., Edelstein, S.J., 1998. Allosteric receptors after 30 years. Neuron 21, 959980. Chavez-Noriega, L.E., Crona, J.H., Washburn, M.S., Urrutia, A., Elliott, K.J., Johnson, E.C., 1997. Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 280, 346356. Corringer, P.J., Le Novere, N., Changeux, J.P., 2000. Nicotinic receptors at the amino acid level. Annu. Rev. Pharmacol. Toxicol. 40, 431458. Dajas-Bailador, F., Wonnacott, S., 2004. Nicotinic acetylcholine receptors and the regulation of neuronal signalling. Trends Pharmacol. Sci. 25, 317324. Darsow, T., Booker, T.K., Pina-Crespo, J.C., Heinemann, S.F., 2005. Exocytic trafficking is required for nicotine-induced

142

B RA I N R E SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

up-regulation of alpha 4 beta 2 nicotinic acetylcholine receptors. J. Biol. Chem. 280, 1831118320. Drago, J., McColl, C.D., Horne, M.K., Finkelstein, D.I., Ross, S.A., 2003. Neuronal nicotinic receptors: insights gained from gene knockout and knockin mutant mice. Cell. Mol. Life Sci. 60, 12671280. Ficklin, M.B., Zhao, S., Feng, G., 2005. Ubiquilin-1 regulates nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. J. Biol. Chem. 280, 3408834095. Fucile, S., 2004. Ca2+ permeability of nicotinic acetylcholine receptors. Cell Calcium 35, 18. Gentry, C.L., Lukas, R.J., 2002. Regulation of nicotinic acetylcholine receptor numbers and function by chronic nicotine exposure. Curr. Drug Targets. CNS Neurol. Disord. 1, 359385. Gotti, C., Clementi, F., 2004. Neuronal nicotinic receptors: from structure to pathology. Prog. Neurobiol. 74, 363396. Gotti, C., Moretti, M., Clementi, F., Riganti, L., McIntosh, J.M., Collins, A.C., Marks, M.J., Whiteaker, P., 2005. Expression of nigrostriatal alpha 6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by beta 3 subunit gene deletion. Mol. Pharmacol. 67, 20072015. Gotti, C., Zoli, M., Clementi, F., 2006. Brain nicotinic acetylcholine receptors: native subtypes and their relevance. Trends Pharmacol. Sci. 27, 482491. Green, W.N., Millar, N.S., 1995. Ion-channel assembly. Trends Neurosci. 18, 280287. Grilli, M., Parodi, M., Raiteri, M., Marchi, M., 2005. Chronic nicotine differentially affects the function of nicotinic receptor subtypes regulating neurotransmitter release. J. Neurochem. 93, 13531360. Halevi, S., Yassin, L., Eshel, M., Sala, F., Sala, S., Criado, M., Treinin, M., 2003. Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression. J. Biol. Chem. 278, 3441134417. Hansen, S.B., Sulzenbacher, G., Huxford, T., Marchot, P., Taylor, P., Bourne, Y., 2005. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24, 36353646. Harkness, P.C., Millar, N.S., 2002. Changes in conformation and subcellular distribution of alpha4beta2 nicotinic acetylcholine receptors revealed by chronic nicotine treatment and expression of subunit chimeras. J. Neurosci. 22, 1017210181. Hogg, R.C., Raggenbass, M., Bertrand, D., 2003. Nicotinic acetylcholine receptors: from structure to brain function. Rev. Physiol., Biochem. Pharmacol. 147, 146. Jeanclos, E.M., Lin, L., Treuil, M.W., Rao, J., DeCoster, M.A., Anand, R., 2001. The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization. J. Biol. Chem. 276, 2828128290. Jensen, A.A., Frolund, B., Liljefors, T., Krogsgaard-Larsen, P., 2005. Neuronal nicotinic acetylcholine receptors: structural revelations, target identifications, and therapeutic inspirations. J. Med. Chem. 48, 47054745. Khiroug, S.S., Harkness, P.C., Lamb, P.W., Sudweeks, S.N., Khiroug, L., Millar, N.S., Yakel, J.L., 2002. Rat nicotinic ACh receptor alpha7 and beta2 subunits co-assemble to form functional heteromeric nicotinic receptor channels. J. Physiol. 540, 425434. Kittler, J.T., Moss, S.J., 2001. Neurotransmitter receptor trafficking and the regulation of synaptic strength. Traffic 2, 437448. Kuryatov, A., Luo, J., Cooper, J., Lindstrom, J., 2005. Nicotine acts as a pharmacological chaperone to up-regulate human alpha4beta2 acetylcholine receptors. Mol. Pharmacol. 68, 18391851. Lai, A., Parameswaran, N., Khwaja, M., Whiteaker, P., Lindstrom, J.M., Fan, H., McIntosh, J.M., Grady, S.R., Quik, M., 2005. Long-term nicotine treatment decreases striatal alpha 6

nicotinic acetylcholine receptor sites and function in mice. Mol. Pharmacol. 67, 16391647. Lansdell, S.J., Gee, V.J., Harkness, P.C., Doward, A.I., Baker, E.R., Gibb, A.J., Millar, N.S., 2005. RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol. Pharmacol. 68, 14311438. Laviolette, S.R., van der Kooy, D., 2004. The neurobiology of nicotine addiction: bridging the gap from molecules to behaviour. Nat. Rev., Neurosci. 5, 5565. Le Novere, N., Corringer, P.J., Changeux, J.P., 2002. The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J. Neurobiol. 53, 447456. Lin, L., Jeanclos, E.M., Treuil, M., Braunewell, K.H., Gundelfinger, E.D., Anand, R., 2002. The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor. J. Biol. Chem. 277, 4187241878. Lindstrom, J., 2000. The structures of neuronal nicotinic receptors. In: Clementi, F., Fornasari, D., Gotti, C. (Eds.), Handbook of Experimental Pharmacology, Vol. Neuronal Nicotinic Receptors. Springer, Berlin, pp. 101162. Marks, M.J., Pauly, J.R., Gross, S.D., Deneris, E.S., Hermans-Borgmeyer, I., Heinemann, S.F., Collins, A.C., 1992. Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J. Neurosci. 12, 27652784. Marks, M.J., Farnham, D.A., Grady, S.R., Collins, A.C., 1993. Nicotinic receptor function determined by stimulation of rubidium efflux from mouse brain synaptosomes. J. Pharmacol. Exp. Ther. 264, 542552. Marks, M.J., Whiteaker, P., Calcaterra, J., Stitzel, J.A., Bullock, A.E., Grady, S.R., Picciotto, M.R., Changeux, J.P., Collins, A.C., 1999. Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit. J. Pharmacol. Exp. Ther. 289, 10901103. Marks, M.J., Rowell, P.P., Cao, J.Z., Grady, S.R., McCallum, S.E., Collins, A.C., 2004. Subsets of acetylcholine-stimulated 86Rb+ efflux and [125I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment. Neuropharmacology 46, 11411157. Moretti, M., Vailati, S., Zoli, M., Lippi, G., Riganti, L., Longhi, R., Viegi, A., Clementi, F., Gotti, C., 2004. Nicotinic acetylcholine receptor subtypes expression during rat retina development and their regulation by visual experience. Mol. Pharmacol. 66, 8596. Moroni, M., Zwart, R., Sher, E., Cassels, B.K., Bermudez, I., 2006. alpha4beta2 nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine. Mol. Pharmacol. 70, 755768. Mugnaini, M., Garzotti, M., Sartori, I., Pilla, M., Repeto, P., Heidbreder, C.A., Tessari, M., 2006. Selective down-regulation of [(125)I]Y0-alpha-conotoxin MII binding in rat mesostriatal dopamine pathway following continuous infusion of nicotine. Neuroscience 137, 565572. Nashmi, R., Dickinson, M.E., McKinney, S., Jareb, M., Labarca, C., Fraser, S.E., Lester, H.A., 2003. Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J. Neurosci. 23, 1155411567. Nelson, M.E., Kuryatov, A., Choi, C.H., Zhou, Y., Lindstrom, J., 2003. Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors. Mol. Pharmacol. 63, 332341. Nguyen, H.N., Rasmussen, B.A., Perry, D.C., 2004. Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment. J. Neurochem. 90, 4049.

B RA I N RE SE A R CH RE V I EW S 55 ( 20 0 7 ) 1 3 41 4 3

143

Palma, E., Maggi, L., Barabino, B., Eusebi, F., Ballivet, M., 1999. Nicotinic acetylcholine receptors assembled from the alpha7 and beta3 subunits. J. Biol. Chem. 274, 1833518340. Parker, S.L., Fu, Y., McAllen, K., Luo, J., McIntosh, J.M., Lindstrom, J.M., Sharp, B.M., 2004. Up-regulation of brain nicotinic acetylcholine receptors in the rat during long-term self-administration of nicotine: disproportionate increase of the alpha6 subunit. Mol. Pharmacol. 65, 611622. Paton, W.D., Zaimis, E.J., 1949. The pharmacological actions of polymethylene bistrimethyl-ammonium salts. Br. J. Pharmacol. Chemother. 4, 381400. Peng, X., Gerzanich, V., Anand, R., Wang, F., Lindstrom, J., 1997. Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y. Mol. Pharmacol. 51, 776784. Perry, D.C., Davila-Garcia, M.I., Stockmeier, C.A., Kellar, K.J., 1999. Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies. J. Pharmacol. Exp. Ther. 289, 15451552. Picciotto, M.R., 2003. Nicotine as a modulator of behavior: beyond the inverted U. Trends Pharmacol. Sci. 24, 493499. Picciotto, M.R., Caldarone, B.J., King, S.L., Zachariou, V., 2000. Nicotinic receptors in the brain. Links between molecular biology and behavior. Neuropsychopharmacology 22, 451465. Picciotto, M.R., Caldarone, B.J., Brunzell, D.H., Zachariou, V., Stevens, T.R., King, S.L., 2001. Neuronal nicotinic acetylcholine receptor subunit knockout mice: physiological and behavioral phenotypes and possible clinical implications. Pharmacol. Ther. 92, 89108. Pidoplichko, V.I., DeBiasi, M., Williams, J.T., Dani, J.A., 1997. Nicotine activates and desensitizes midbrain dopamine neurons. Nature 390, 401404. Quik, M., Vailati, S., Bordia, T., Kulak, J.M., Fan, H., McIntosh, J.M., Clementi, F., Gotti, C., 2005. Subunit composition of nicotinic receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA. Mol. Pharmacol. 67, 3241. Ren, X.Q., Cheng, S.B., Treuil, M.W., Mukherjee, J., Rao, J., Braunewell, K.H., Lindstrom, J.M., Anand, R., 2005. Structural determinants of alpha4beta2 nicotinic acetylcholine receptor trafficking. J. Neurosci. 25, 66766686. Riganti, L., Matteoni, C., Di Angelantonio, S., Nistri, A., Gaimarri, A., Sparatore, F., Canu-Boido, C., Clementi, F., Gotti, C., 2005. Long-term exposure to the new nicotinic antagonist 1,2-bisN-cytisinylethane upregulates nicotinic receptor subtypes of SH-SY5Y human neuroblastoma cells. Br. J. Pharmacol. 146, 10961109. Rowell, P.P., Wonnacott, S., 1990. Evidence for functional activity of up-regulated nicotine binding sites in rat striatal synaptosomes. J. Neurochem. 55, 21052110.

Sallette, J., Bohler, S., Benoit, P., Soudant, M., Pons, S., Le Novere, N., Changeux, J.P., Corringer, P.J., 2004. An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine. J. Biol. Chem. 279, 1876718775. Sallette, J., Pons, S., Devillers-Thiery, A., Soudant, M., Prado de Carvalho, L., Changeux, J.P., Corringer, P.J., 2005. Nicotine upregulates its own receptors through enhanced intracellular maturation. Neuron 46, 595607. Sine, S.M., Engel, A.G., 2006. Recent advances in Cys-loop receptor structure and function. Nature 440, 448455. Tumkosit, P., Kuryatov, A., Luo, J., Lindstrom, J., 2006. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6 nicotinic acetylcholine receptors expressed in transfected cell lines. Mol. Pharmacol. 70, 13581368. Vallejo, Y.F., Buisson, B., Bertrand, D., Green, W.N., 2005. Chronic nicotine exposure upregulates nicotinic receptors by a novel mechanism. J. Neurosci. 25, 55635572. Villarroya, M., Gandia, L., Lopez, M.G., Garcia, A.G., Cueto, S., Garcia-Navio, J.L., Alvarez-Builla, J., 1996. Synthesis and pharmacology of alkanediguanidinium compounds that block the neuronal nicotinic acetylcholine receptor. Bioorg. Med. Chem. 4, 11771183. Vincler, M., Wittenauer, S., Parker, R., Ellison, M., Olivera, B.M., McIntosh, J.M., 2006. Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. U. S. A. 103, 1788017884. Wang, F., Nelson, M.E., Kuryatov, A., Olale, F., Cooper, J., Keyser, K., Lindstrom, J., 1998. Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells. J. Biol. Chem. 273, 2872128732. Williams, B.M., Temburni, M.K., Levey, M.S., Bertrand, S., Bertrand, D., Jacob, M.H., 1998. The long internal loop of the alpha 3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo. Nat. Neurosci. 1, 557562. Williams, M.E., Burton, B., Urrutia, A., Shcherbatko, A., Chavez-Noriega, L.E., Cohen, C.J., Aiyar, J., 2005. Ric-3 promotes functional expression of the nicotinic acetylcholine receptor alpha7 subunit in mammalian cells. J. Biol. Chem. 280, 12571263. Xu, J., Zhu, Y., Heinemann, S.F., 2006. Identification of sequence motifs that target neuronal nicotinic receptors to dendrites and axons. J. Neurosci. 26, 97809793. Yu, Z.J., Wecker, L., 1994. Chronic nicotine administration differentially affects neurotransmitter release from rat striatal slices. J. Neurochem. 63, 186194.