Nicotine and Alpha3beta2 Neuronal Nicotinic Acetylcholine Receptors

C H A P T E R

30 Nicotine and Alpha3beta2 Neuronal Nicotinic Acetylcholine Receptors Doris Clark Jackson, Sterling N. Sudweeks Brigham Young University, Physiology and Developmental Biology, Provo, UT, United States

the homomeric α7 and the α4β2. We have characterized at least two subtypes of the α3β2 nAChR (Fig. 30.2) and will detail the possible role the α3β2 nAChRs play in nicotine addiction.

Abbreviations ACh EC50 HEK-293 M1–M4 nAChR SNP VTA

acetylcholine effective concentration for 50% activation human embryonic kidney cells transmembrane region nicotinic acetylcholine receptor single-nucleotide polymorphism ventral tegmental area

30.2 NICOTINE SENSITIVITY BY SUBTYPE

30.1 INTRODUCTION Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated ion channels found both neuronally and at the neuromuscular junction. Each subunit consists of four transmembrane regions (M1–M4) with the intracellular M3–M4 loop being the least conserved regions between subunits. The M2 of each subunit forms the receptor’s pore (Fig. 30.1). nAChRs are cation-selective with some subtypes permeable to Ca2+ (α3β2 are somewhat permeable; inclusion of α5 significantly increases Ca2+ permeability). Ca2+-permeable nAChRs play the unique role of facilitating and even independently stimulating the release of other neurotransmitters like dopamine when these receptors are found presynaptically. The nAChR found at the neuromuscular junction is made of five different subunits (α1, β1, γ, δ, and ε). Neuronal nAChRs can be both homomeric and heteromeric. However, only the α7 and α9 subunits can form homomeric receptors. Most neuronal nAChR are a combination of α and β subunits (α2, α3, α4, α5 (must have an additional α subunit), α6, α7, α8 (chicken), α9, α10, β2, β3, and β4). The most common heteromeric nAChRs are those with two α2 and three βs (α*2β*3) or three αs and two β2 (α*3β*2). The most characterized nAChR subtypes are

Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00030-7

The α3β2 nAChRs are shown to be one of the least nicotine sensitive nAChRs subtypes and only a partial agonist of nicotine (Gerzanich, Wang, Kuryatov, & Lindstrom, 1998; Olale, Gerzanich, Kuryatov, Wang, & Lindstrom, 1997; Wang et al., 1996, 1998). Traditionally, only the α4β2 nAChRs were considered high-affinity nicotine binding sites. However, the α4β2 nAChRs only represent 90% of the high-affinity nicotine binding sites. Both the α and β subunits contribute to the nicotine affinity and efficacy. The β2 subunit has been shown to have greater nicotine binding affinity and more easily desensitized when compared to the β4 subunit (Fenster, Rains, Noerager, Quick, & Lester, 1997; Parker, Beck, & Luetje, 1998), whereas α3* receptors are less desensitized than α4* receptors (Fenster et al., 1997). Likewise, inclusion of the α5 subunit in the α3β2 increases nicotine efficacy significantly (Wang et al., 1996, 1998). However, this is unlikely due to a change in nicotine affinity but in channel gating (Wang et al., 1998). The most likely location of the α5 subunit would not change the binding site but instead impact the channel lining (Wang et al., 1996). Differences in amino acids of M1 (224 and 226) account for the higher nicotine affinity of the β2 subunit as compared to the β4 (Rush, Kuryatov, Nelson, & Lindstrom, 2002). Differences in the amino acids at position 226 of

235

Copyright © 2019 Elsevier Inc. All rights reserved.

236

30. NICOTINE AND ALPHA3BETA2 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

FIG. 30.1 nAChR subunit and embedded assembly. (A) Each nicotinic acetylcholine receptor (nAChR) subunit contains four transmembrane regions. The intracellular loop between transmembrane regions 3 and 4 (M3–M4) is the most variable between nAChR subunits. (B) Five subunits (usually a combination of αs and βs) form a functional receptor with M2 forming the channel pore.

Normalized peak current

1.25 1.00 0.75 0.50 0.25 0.00 –8 –0.25

–7

–6

–5

–4

–3

–2

–1

[ACh] 1:5

5:1

FIG. 30.2 ACh dose-response curves differentiate α3β2 subtypes. Upon injection of α3 and β2 human mRNA into X. laevis oocytes in 1:5 and 5:1 ratios, two distinct populations of nicotinic acetylcholine receptors (nAChRs) arise. More β and less α expression resulted in more acetylcholine (ACh)-sensitive α3β2 subtypes (EC50 ¼ 12.2  1.7 μM; nH ¼ 0.49  0.13 (R2 ¼ 0.74)) (n ¼ 14, replicates of 4, 1 outlier removed), whereas more β and less α expression resulted in less ACh-sensitive α3β2 subtypes (EC50 ¼ 263.8  1.6 μM; nH ¼ 0.55  0.15 (R2 ¼ 0.77)) (n ¼ 12, replicates of 4, 2 outliers removed). One-way ANOVA analysis resulted in significant differences (F[15, 258] ¼ 54.644; ***P < .001). Data represented are mean  SEM.

M1 (Fig. 30.1) account for the difference in α3* and α4* nAChR subtype nicotine binding affinity (Rush et al., 2002). Even though the α3 has greater binding affinity than α4, the α3β2 nAChR only has partial efficacy. Rush et al. (2002) suggest several mechanisms for the partial agonist activity of nicotine on α3β2 nAChRs. One mechanism may be that nicotine binding does not effectively shift the channel to the open state. Another mechanism for the partial agonist activity of nicotine may be that nicotine only weakly binds to the binding site. Kuryatov, Olale, Cooper, Choi, and Lindstrom (2000) showed that when using a chimera that consisted of the α4β2 binding site with the α3β2 channel, nicotine acts as a partial agonist; whereas, when using the α3β2

binding site with the α4β2 channel nicotine, had full efficacy. Their conclusion was that the α3β2 nAChRs have channel block when activated with nicotine. A study by Rush et al. (2002) supports the claim that nicotine may cause a channel block through binding to a low-affinity binding site that occludes the channel. However, the concentration of nicotine required for channel blocking is larger than would be expected by smokers (Benowitz, Porchet, & Jacob, 1990). Additionally, when an α6 subunit is incorporated into the α3β2 nAChR, nicotine has 100% efficacy (Kuryatov et al., 2000). Therefore, multiple α3β2 interfaces may be required for the channel block (Rush et al., 2002).

30.3 NICOTINE-INDUCED UPREGULATION Like other nAChR subtypes, the α3β2 nAChR is upregulated upon nicotine exposure. General nAChR upregulation is observed in rat, mouse, and postmortem human brains (Breese et al., 1997; Perry, Davila-Garcia, Stockmeier, & Kellar, 1999; Yates, Bencherif, Fluhler, & Lippiello, 1995). Like nicotine binding affinity, subunit composition affects different phases of nicotine-induced upregulation. For example, β2* nAChRs have a much larger rate of nicotine-induced upregulation than β4* containing nAChRs (Gahring, Osborne-Hereford, Vasquez-Opazo, & Rogers, 2008; Wang et al., 1998). However, Meyer, Xiao, and Kellar (2001) did not show a change in upregulation with the addition of β4. When comparing three subtypes, the α3β2, α4β2, and α6β2, the α3β2 receptor required the highest nicotine concentration for upregulation and was about 10 times faster than the α4β2 upregulation but slightly slower than the α6β2 nAChR upregulation (Walsh et al., 2008). Therefore, the α3β2 nAChR may do little during initial nicotine exposure but play a more significant role in chronic exposure if nicotine levels are high. Nicotine upregulation of α3β2 receptors occurs in human embryonic kidney cells (HEK-293) and human neuroblastoma SH-SY5Y, as well as neurons as evidenced by postmortem brains, indicating that upregulation is dependent on the receptor not the response of the neuron cellular processes (Wang et al., 1998). However, Wang et al. (1998) did not show upregulation in Xenopus laevis oocytes. The X. laevis oocytes may not have the cellular machinery required, like Rapsyn, to allow for upregulation. Nonetheless, the human cell lines and neurons are more directly related to human physiology than X. laevis oocytes. This should be considered when interpreting data regarding nAChR upregulation in oocytes. Upregulation is likely both pre- and posttranscriptional. In HEK-293 cells (tsA201), chronic nicotine exposure (for upregulation, EC50 ¼ 2  0.3 μM; for nicotine

237

30.4 LOCATION

activation, EC50 ¼ 70  6 μM) resulted in a 24-fold increase in α3β2 nAChRs (as well as the α3β2α5). Upregulation was observed as early as 15 min following initial nicotine exposure but continued to increase with prolonged exposure (Wang et al., 1998). It is important to note that the nicotine concentration required for upregulation is much lower and physiologically more similar to serum nicotine levels in smokers than simply the nicotine level required for α3β2 receptor activation. The change was noted as an increase in cell surface expression and an increase in the total number of nAChRs. Upregulation that began almost immediately would likely indicate increased trafficking and increased assembly. In addition, to an increase in receptor membrane integration, the endoplasmic reticulum slows the degradation of the α3β2 nAChR resulting in an increase in the overall receptor expression. Specifically, Rezvani et al. (2009) showed that nicotine can prevent protein degradation by partially inhibiting the ubiquitin proteasome system. As a note, chronic nicotine exposure to the human neuroblastoma SH-SY5Y cell line resulted in only a sixfold increase in α3β2 nAChRs. Yet, this was simply an increase in receptor number, not membrane integration. Interestingly, chronic nicotine exposure in the HEK-293 and the human neuroblastoma SH-SY5Y did not result in upregulation of either the α3β4 or the α3β4α5 (Wang et al., 1998). In addition, temperature and pro-inflammatory cytokines can influence nicotine-induced upregulation complicating the analysis and interpretation of results. TNF-α increases α3β2 upregulation during transcription and translational phases although to a smaller degree than α4β2 and α4β4 (Gahring et al., 2008). Inflammation caused by smoking may further compound the effects of smoking. In general, lowering the temperature decreases receptor turnover that may influence results for upregulation giving a falsely high result (Devreotes & Fambrough, 1975; Paulson & Claudio, 1990). For the α4β2 nAChR, lowering the temperature to 29°C or 30°C significantly increases the cell surface expression (Cooper, Harkness, Baker, & Millar, 1999). This is an interesting effect considering that cigarette smoking causes a 1.5°C drop in skin temperature (Benowitz, Jacob III, & Herrera, 2006). Upregulation can also be impacted through changes in phosphorylation. For example, the muscle nAChR is upregulation via cAMP and protein kinase A. This mechanism increases assembly efficiency and prevents degradation (Green, Ross, & Claudio, 1991). Wang et al. (1998) show that H-7, a protein kinase inhibitor, blocks at least 50% of nicotine-induced upregulation of α3β2 nAChRs in HEK-293 cells (tsA201). Therefore, there are many likely factors in the cellular process for nicotine upregulation including subunit composition, cell expression system, temperature, cytokines, and phosphorylation.

30.4 LOCATION When considering the location of α3β2 nAChRs, the limiting subunit is the α3. The β2 subunit is quite ubiquitous, at least in the central nervous system, but is also readily found in the peripheral nervous system (Fig. 30.3, Hill Jr, Zoli, Bourgeois, & Changeux, 1993). In the rat and mouse, the α3 subunit is found in the thalamus, medial habenula, superior colliculus, and pineal body (Fig. 30.4, Cimino, Marini, Fornasari, Cattabeni, & Clementi, 1992). With a more limited expression pattern, the α3 subunit is more likely to be the limiting subunit in α3β2 expression (Fig. 30.5). The β2 knockout mice do not self-administer nicotine, and the mesolimbic neurons do not release dopamine in response to nicotine virtually abolishing addiction (Picciotto et al., 1998), whereas the α3 knockout mice results in multiple autonomic system problems (Xu et al., 1999). In addition to these locations, the Macaca fascicularis (cynomolgus monkey) identifies the α3 subunit in the hippocampus (Cimino et al., 1992). This difference may be important in translating research to humans considering the much higher conservation between humans and nonhuman primates (Shorey-Kendrick,

FIG. 30.3 β2 nAChR expression in the mouse brain.

Heat map (red high, blue low) detailing the ubiquitous β2 subunit expression in the mouse brain. The expression profile is quantified in Fig. 30.5. Image: With permission: Allen Brain Atlas, http://mouse.brainmap.org/experiment/show/2098.

238

30. NICOTINE AND ALPHA3BETA2 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

Alpha 3

Beta 2

Isocortex Olfactory areas

0.19 0.19

2.96 3.37

Hippocampal formation Cortical subplate Striatum Pallidum

0.12 0.03 0.07 0.08

4.23 3.53 1.93 3

2015). Additionally, both the M. fascicularis and in human tissue identify the α3 mRNA subunit at high levels in sympathetic and parasympathetic ganglia (Cimino et al., 1992). The location of the α3 subunit will likely dictate the presence of α3β2 nAChRs in the central nervous system, and the β2 subunit will likely dictate α3β2 expression in the peripheral nervous system. In the central nervous system, α3β2 nAChRs influence striatal dopamine release and eventual addiction (Picciotto et al., 1998). However, the α3β4 nAChRs are shown to contribute more significantly than the α3β2 nAChRs in the peripheral nervous system (Covernton, Kojima, Sivilotti, Gibb, & Colquhoun, 1994). Yet, the α3 subunit remains the predominant subunit in the peripheral nervous system. Therefore, the α3β2 nAChRs may play a larger role in the autonomic physiological nicotine response. The dopamine neurons of the midbrain (superior colliculus) likely contribute to the physical locomotion required for nicotine self-administration. Blocking dopamine receptors prevents nicotine self-administration (Corrigall & Coen, 1991). With a moderately high expression of the α3 subunit in the superior colliculus, the α3β2 nAChR may impact dopamine release and indirectly effect nicotine self-administration. Additionally, the α3 and β2 mRNA subunits have been identified in the ventral tegmental area (VTA) and the nucleus accumbens using high-affinity nicotine and n-bungarotoxin binding (Azam, Winzer-Serhan, Chen, & Leslie, 2002). The α3β2 nAChR has not been identified as the most important contributor to nicotine addiction in the VTA, but it may contribute to a different stage in addiction considering its upregulation and increased nicotine affinity following nicotine exposure as compared to α4β2 (Azam et al., 2002). Autoradiography and experiments using 125I-nBgt or 125I-α-conotoxin MII label nAChRs at the presynaptic dopaminergic neurons of the nucleus accumbens. Although α-conotoxin MII was originally thought to be α3β2 specific, it has since been shown to bind α6* nAChRs as well (Kuryatov et al., 2000; Parker et al., 2004). Therefore, either/both α3 and α6 containing nAChRs may contribute to dopamine release in the nucleus accumbens. Furthermore, the α3 subunit has relatively high levels in the habenula (Fig. 30.4) and therefore may additionally mediate dopamine release in this pathway and contribute to the nicotine-addicted state.

Thalamus Hypothalamus Midbrain Pons Medulla Cerebellum

0.37 0.14 0.63 0.14 0.18 0.15

4.76 3.84 3.12 2.51 2.38 2.97

30.5 PHYSIOLOGICAL EFFECT OF NICOTINE

FIG. 30.4 α3 nAChR expression in the mouse brain. Heat map (red high, blue low) detailing the limited α3 subunit expression in the mouse brain. Lateral habenula (pink), olivary pretectal nucleus (purple), and superior colliculus (green). Image: With permission: Allen Brain Atlas, http://mouse.brain-map.org/experiment/show/69734723.

Cerebellum Medulla Pons Midbrain Hypothalamus Thalamus Pallidum Striatum Cortical subplate Hippocampal formation Olfactory areas Isocortex 0

1

2 Beta 2

Mouse raw expression values from Allen Brain Atlas

3

4

5

Alpha 3

FIG. 30.5 Mouse raw expression values from Allen Brain Atlas. The relative expression profile of mouse (C57BL/6J) brain regions containing both α3 and β2 subunits. Results were obtained using antisense in situ hybridization of an adult (56 days) male mouse. As indicated, the β2 subunit has higher expression than the α3 in all regions analyzed. Data from Allen Brain Atlas with permission.

The nicotine dose-response curve for the α3β2 nAChRs expressed in HEK 293 cells with a 1:1 α3-β2 ratio by Wang et al. (1998) has a nicotine EC50 ¼ 70  26 μM, which is higher than would be expected for blood nicotine levels

239

30.8 CONCLUSION

for smokers. Nicotine serum concentration is 0.2 μM for a typical smoker (Benowitz et al., 1990). Therefore, the α3β2 nAChRs may not play a role in initial nicotine addiction. However, considering the high upregulation of α3β2 nAChRs following nicotine exposure, α3β2 nAChRs may be important in sustaining addiction. The α4β2 nAChR is highly sensitive to nicotine and may play an important role in initial addiction. At typical nicotine serum levels, the α4β2 nAChRs would be upregulated like the α3β2 nAChRs, but they would also be almost fully desensitized. However, α3β2 nAChRs (not specific stoichiometries) are not as easily desensitized and would likely maintain normal functioning in smokers as they remain 80% active at normal serum nicotine level in smokers (Fenster et al., 1997; Olale et al., 1997; Picciotto et al., 1998). The α3β2 nAChRs may contribute more significantly to acetylcholine (ACh)-mediated signaling in a nicotine-addicted brain than the α4β2 nAChRs (Olale et al., 1997; Wang et al., 1998). Furthermore, the upregulation or lack thereof may vary between brain regions just as there was variability between cell lines (HEK-293, neuroblastoma, and oocytes) (Wang et al., 1998). This possible and likely variability in upregulation would likely influence behavior considering that brain regions are affected more than others. However, none of these upregulation studies were done on specific α3β2 nAChR subtypes. Our preliminary data, like that of other nAChR subtypes, show a difference in nicotine affinity and efficacy between different stoichiometries of the α3β2 nAChRs. The α3 nAChR is by far the most populous subunit in the autonomic nervous system. Although the predominant nAChR in the autonomic ganglia is the α3β4 (Vernallis, Conroy, & Berg, 1993), the α3β2 likely also plays a significant role in signaling and nicotine response. Nicotine has many effects on the autonomic nervous system including increased heart rate, increased myocardial contractility, increased respiration, constriction of arteries, sweating, nausea, diarrhea, and increased blood pressure (Haass & K€ ubler, 1997).

30.6 VARIANTS OF THE α3 SUBUNIT The α3 nAChR subunit has single-nucleotide variants (rs3743078, rs6495308, and rs1051730) associated with increased nicotine cravings, increased smoking, or increased nicotine-related behaviors (Shmulewitz et al., 2016; Wu et al., 2015). Nees et al. (2013) conclude that the single-nucleotide polymorphism (SNP) rs578776 found on the α3 gene is associated with increased risk of smoking because it dampens the reward response of the anterior cingulate cortex. Several of these polymorphisms are included in a gene cluster between the α5-α 3-β4 subunits that is associated with nicotine dependence.

A study by Polina et al. (2014) shows that two polymorphisms (rs578776 and rs3743078) in the α3 gene are associated with an increased risk of smoking in patients with ADHD but are protective for smoking in non-ADHD populations. Additionally, there multiple polymorphisms (rs578776, rs938682, rs6495309, and rs3743073) identified in the α3 nAChR subunit that increase susceptibility to lung cancer (Qu et al., 2016; Shen et al., 2012; Xiao, Chen, Wu, & Wen, 2014). Two other polymorphisms (rs8042059 and rs7177514) in the α3 nAChR subunit likely increase susceptibility to lung cancer only indirectly through smoking behavior (Zhou et al., 2015). The SNPs rs12910984, rs6495309, and rs1051730 have been shown to be associated with an increased risk of chronic obstructive pulmonary disease (Kaur-Knudsen, Nordestgaard, & Bojesen, 2012; Kim et al., 2013; Yang et al., 2012). The SNP rs6495308 has been found to be associated with an increased risk of hypertension (Wu et al., 2015). The rs8042374 a SNP on the α3 subunit is associated with increased risk of adenocarcinoma (He et al., 2014).

30.7 IMPLICATIONS FOR TREATMENT As highlighted, variants in the α3 subunit may affect the likelihood of addiction and likelihood of nicotinerelated diseases and even help predict response to treatment. By identifying and characterizing the effects of these variants in various populations, nicotine addiction treatment may prove to be more personalized and more effective. We are still in the early stages of characterizing the α3β2 nAChR and its relation to nicotine addiction, but the evidence suggests that by targeting the α3β2, we may be able to counteract the autonomic effects of nicotine considering the location of the α3 subunit. Additionally, considering the location and function of the α3β2 nAChR, pharmacological approaches to treatment targeting the α3β2 nAChR may alter the dopamine release associated with addiction and therefore make nicotine less pleasurable. Lastly, since pro-inflammatory cytokines result in the upregulation of the α3β2 nAChR and other subtypes, the use of antiinflammatory drugs or diet should be considered when developing combinatory treatment options.

30.8 CONCLUSION In summary, the α3β2 nAChR may play a role in both central and peripheral nervous system nicotine responses. The α3β2 nAChR is upregulated upon nicotine exposure and has many variants that can influence the addictive and disease effects of nicotine.

240

30. NICOTINE AND ALPHA3BETA2 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

MINI-DICTIONARY OF TERMS Affinity Affinity refers to the concentration required for receptor activation. A nicotine higher affinity means a smaller concentration of nicotine is required for activation, whereas a low nicotine affinity means a larger concentration of nicotine is required for activation. Efficacy Efficacy refers to the maximum activation that can be reached. Efficacy can only be compared across subtypes or agonists. Nicotine-induced upregulation In this context, nicotine-induced upregulation refers to an increase in the number of nAChRs expressed following nicotine exposure. Upregulation may alter the cell response to chronic nicotine exposure. Nicotinic acetylcholine receptor (nAChR) A neurotransmitter receptor that conducts positive ions resulting in a change in electric potential. This change in electric potential may induce cell-to-cell communication between neurons or other cell types. Transmembrane region The region of the nAChR protein that spans the membrane. The transmembrane region is mostly hydrophobic and helps form the channel pore. nAChR subunits have four transmembrane regions. Variants Variants are small differences in subunit proteins. These differences may result from changes in the gene sequence or protein production.

Key Facts of Neuronal Nicotinic Acetylcholine Receptors • Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated ion channels that are activated using the endogenous neurotransmitter acetylcholine or the exogenous chemical nicotine. • Most neuronal nAChRs are made of α and β subunits. • Neuronal nAChRs are cation-selective with specific subtypes being calcium (Ca2+) permeable. • Neuronal nAChRs can be located presynaptically, postsynaptically, periterminally, and even on nonneuron cells. • Depending on location and Ca2+ permeability, specific subtypes can induce neurotransmitter release. • Nicotine upregulates and desensitizes many subtypes of neuronal nAChRs. • nAChR subunit variants may alter nicotine sensitivity or physiological response to nicotine. Summary Points • The α3β2 neuronal nicotinic acetylcholine receptor (nAChR) and its relationship to nicotine and nicotine addiction. • The α3β2 neuronal nAChR is only a partial nicotine agonist and requires a higher nicotine concentration for activation as compared to other subtypes. • The α3β2 neuronal nAChR is significantly upregulated more than the α4β2 nAChR. • The α3β2 neuronal nAChR remains 80% active at nicotine concentrations that desensitize other nAChR subtypes.

• The α3 subunit is the most prominent nAChR subtype in the autonomic ganglia. • Nicotine affects many aspects of the autonomic nervous system including heart rate, blood pressure, and digestion. • The location of the α3 subunit likely dictates the presence of α3β2 nAChRs in the central nervous system, whereas the location of the β2 subunit likely dictates the presence of the α3β2 nAChRs in the autonomic ganglia. • Several variants of the α3 subunit increase the likelihood of nicotine addiction and/or the susceptibility to nicotine-related diseases.

References Azam, L., Winzer-Serhan, U. H., Chen, Y., & Leslie, F. M. (2002). Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. The Journal of Comparative Neurology, 444, 260–274. Benowitz, N. L., Jacob, P., III, & Herrera, B. (2006). Nicotine intake and dose response when smoking reduced nicotine content cigarettes. Clinical Pharmacology and Therapeutics, 80, 703–714. Benowitz, N., Porchet, H., & Jacob, P. (1990). In S. Wonnacott, M. Russell, & I. Stolerman (Eds.), Nicotine psychopharmacology (pp. 112–157). Oxford, England: Oxford University. Breese, C. R., Marks, M. J., Logel, J., Adams, C. E., Sullivan, B., Collins, A. C., et al. (1997). Effect of smoking history on [3H]nicotine binding in human postmortem brain. The Journal of Pharmacology and Experimental Therapeutics, 282, 7–13. Cimino, M., Marini, P., Fornasari, D., Cattabeni, F., & Clementi, F. (1992). Distribution of nicotinic receptors in cynomolgus monkey brain. Neuroscience, 51, 77–86. Cooper, S. T., Harkness, P. C., Baker, E. R., & Millar, N. S. (1999). Upregulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits. The Journal of Biological Chemistry, 274, 27145–27152. Corrigall, W. A., & Coen, K. M. (1991). Selective dopamine antagonists reduce nicotine self-administration. Psychopharmacology, 104, 171–176. Covernton, P. J., Kojima, H., Sivilotti, L. G., Gibb, A. J., & Colquhoun, D. (1994). Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes. The Journal of Physiology, 481(Pt1), 27–34. Devreotes, P. N., & Fambrough, D. M. (1975). Acetylcholine receptor turnover in membranes of developing muscle fibers. The Journal of Cell Biology, 65, 335–358. Fenster, C. P., Rains, M. F., Noerager, B., Quick, M. W., & Lester, R. A. (1997). Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine. The Journal of Neuroscience, 17, 5747–5759. Gahring, L. C., Osborne-Hereford, A. V., Vasquez-Opazo, G. A., & Rogers, S. W. (2008). Tumor necrosis factor alpha enhances nicotinic receptor up-regulation via a p38MAPK-dependent pathway. The Journal of Biological Chemistry, 283, 693–699. Gerzanich, V., Wang, F., Kuryatov, A., & Lindstrom, J. (1998). Alpha 5 subunit alters desensitization pharmacology Ca++ permeability and Ca ++ modulation of human neuronal alpha 3 nicotinic receptors. The Journal of Pharmacology and Experimental Therapeutics, 286, 311–320.

REFERENCES

Green, W. N., Ross, A. F., & Claudio, T. (1991). Acetylcholine receptor assembly is stimulated by phosphorylation of its gamma subunit. Neuron, 7, 659–666. Haass, M., & K€ ubler, W. (1997). Nicotine and sympathetic neurotransmission. Cardiovascular Drugs and Therapy, 10, 657–665. He, P., Yang, X. X., He, X. Q., Chen, J., Li, F. X., Gu, X., et al. (2014). CHRNA3 polymorphism modifies lung adenocarcinoma risk in the Chinese Han population. International Journal of Molecular Sciences, 15, 5446–5457. Hill, J. A., Jr., Zoli, M., Bourgeois, J. P., & Changeux, J. P. (1993). Immunocytochemical localization of a neuronal nicotinic receptor: the beta 2-subunit. The Journal of Neuroscience, 13(4), 1551–1568. Kaur-Knudsen, D., Nordestgaard, B. G., & Bojesen, S. E. (2012). CHRNA3 genotype, nicotine dependence, lung function and disease in the general population. The European Respiratory Journal, 40, 1538–1544. Kim, W. J., Oh, Y. M., Kim, T. H., Lee, J. H., Kim, E. K., Lee, J. H., et al. (2013). CHRNA3 variant for lung cancer is associated with chronic obstructive pulmonary disease in Korea. Respiration, 86, 117–122. Kuryatov, A., Olale, F., Cooper, J., Choi, C., & Lindstrom, J. (2000). Human alpha6 AChR subtypes: subunit composition, assembly and pharmacological responses. Neuropharmacology, 39, 2570–2590. Meyer, E. L., Xiao, Y., & Kellar, K. J. (2001). Agonist regulation of rat alpha 3 beta 4 nicotinic acetylcholine receptors stably expressed in human embryonic kidney 293 cells. Molecular Pharmacology, 60, 568–576. Nees, F., Witt, S. H., Lourdusamy, A., Vollst€adt-Klein, S., Steiner, S., Poustka, L., et al. (2013). Genetic risk for nicotine dependence in the cholinergic system and activation of the brain reward system in healthy adolescents. Neuropsychopharmacology, 38(11), 2081–2089. Olale, F., Gerzanich, V., Kuryatov, A., Wang, F., & Lindstrom, J. (1997). Chronic nicotine exposure differentially affects the function of human alpha3, alpha4, and alpha7 neuronal nicotinic receptor subtypes. The Journal of Pharmacology and Experimental Therapeutics, 283, 675–683. Parker, M. J., Beck, A., & Luetje, C. W. (1998). Neuronal nicotinic receptor beta2 and beta4 subunits confer large differences in agonist binding affinity. Molecular Pharmacology, 54(6), 1132–1139. Parker, S. L., Fu, Y., McAllen, K., Luo, J., McIntosh, J. M., Lindstrom, J. M., et al. (2004). Upregulation of brain nicotinic acetylcholine receptors in the rat during long-term self-administration of nicotine: disproportionate increase of the alpha6 subunit. Molecular Pharmacology, 65, 611–622. Paulson, H. L., & Claudio, T. (1990). Temperature-sensitive expression of all-Torpedo and Torpedorat hybrid AChR in mammalian muscle cells. The Journal of Cell Biology, 110, 1705–1717. 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. The Journal of Pharmacology and Experimental Therapeutics, 289, 1545–1552. Picciotto, M. R., Zoli, M., Rimondini, R., Lena, C., Marubio, L. M., Pich, E. M., et al. (1998). Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature, 391, 173–177. Polina, E. R., Rovaris, D. L., de Azeredo, L. A., Mota, N. R., Vitola, E. S., Silva, K. L., et al. (2014). ADHD diagnosis may influence the association between polymorphisms in nicotinic acetylcholine receptor genes and tobacco smoking. Neuro Molecular Medicine, 16, 389–397. Qu, X., Wang, K., Dong, W., Shen, H., Wang, Y., Liu, Q., et al. (2016). Association between two CHRNA3 variants and susceptibility of lung cancer: a meta-analysis. Scientific Reports, 6, 20149.

241

Rezvani, K., Teng, Y., Pan, Y., Dani, J. A., Lindstrom, J., García Gras, E. A., et al. (2009). UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors. The Journal of Neuroscience, 29(21), 6883–6896. Rush, R., Kuryatov, A., Nelson, M. E., & Lindstrom, J. (2002). First and second transmembrane segments of alpha3, alpha4, beta2, and beta4 nicotinic acetylcholine receptor subunits influence the efficacy and potency of nicotine. Molecular Pharmacology, 61, 1416–1422. Shen, B., Shi, M. Q., Zheng, M. Q., Hu, S. N., Chen, J., & Feng, J. F. (2012). Correlation between polymorphisms of nicotine acetylcholine acceptor subunit CHRNA3 and lung cancer susceptibility. Molecular Medicine Reports, 6, 1389–1392. Shmulewitz, D., Meyers, J. L., Wall, M. M., Aharonovich, E., Frisch, A., Spivak, B., et al. (2016). CHRNA5/A3/B4 variant rs3743078 and nicotine-related phenotypes: indirect effects through nicotine craving. Journal of Studies on Alcohol and Drugs, 77(2), 227–237. Shorey-Kendrick, L. E., Ford, M. M., Allen, D. C., Kuryatov, A., Lindstrom, J., Wilhelm, L., et al. (2015). Nicotinic receptors in nonhuman primates: Analysis of genetic and functional conservation with humans. Neuropharmacology, 96(Pt B), 163–173. Vernallis, A. B., Conroy, W. G., & Berg, D. K. (1993). Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes. Neuron, 10(3), 451–464. Walsh, H., Govind, A. P., Mastro, R., Hoda, J. C., Bertrand, D., Vallejo, Y., et al. (2008). Upregulation of nicotinic receptors by nicotine varies with receptor subtype. The Journal of Biological Chemistry, 283, 6022–6032. Wang, F., Gerzanich, V., Wells, G. B., Anand, R., Peng, X., Keyser, K., et al. (1996). Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2 and beta4 subunits. The Journal of Biological Chemistry, 271, 17656–17665. Wang, F., Nelson, M. E., Kuryatov, A., Olale, F., Cooper, J., Keyser, K., et al. (1998). Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells. The Journal of Biological Chemistry, 273, 28721–28732. Wu, X. Y., Zhou, S. Y., Niu, Z. Z., Liu, T., Xie, C. B., & Chen, W. Q. (2015). CHRNA3 rs6495308 genotype as an effect modifier of the association between daily cigarette consumption and hypertension in Chinese male smokers. International Journal of Environmental Research and Public Health, 12, 4156–4169. Xiao, M., Chen, L., Wu, X., & Wen, F. (2014). The association between the rs6495309 polymorphism in CHRNA3 gene and lung cancer risk in Chinese: a meta-analysis. Scientific Reports, 4, 6372. Xu, W., Orr-Urtreger, A., Nigro, F., Gelber, S., Sutcliffe, C. B., Armstrong, D., et al. (1999). Multiorgan autonomic dysfunction in mice lacking the beta2 and the beta4 subunits of neuronal nicotinic acetylcholine receptors. The Journal of Neuroscience, 19, 9298–9305. Yang, L., Qiu, F., Lu, X., Huang, D., Ma, G., & Guo, Y. (2012). Functional polymorphisms of CHRNA3 predict risks of chronic obstructive pulmonary disease and lung cancer in Chinese. PLoS ONE, 7(10). e46071. Yates, S. L., Bencherif, M., Fluhler, E. N., & Lippiello, P. M. (1995). Upregulation of nicotinic acetylcholine receptors following chronic exposure of rats to mainstream cigarette smoke or alpha 4 beta 2 receptors to nicotine. Biochemical Pharmacology, 50, 2001–2008. Zhou, W., Geng, T., Wang, H., Xun, X., Feng, T., Zou, H., et al. (2015). CHRNA3 genetic polymorphism and the risk of lung cancer in the Chinese Han smoking population. Tumour Biology, 36, 4987–4992.