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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Aug 8, 2012.
Published in final edited form as:
PMCID: PMC3286518

Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms


Nicotine causes changes in brain nicotinic acetylcholine receptors (nAChRs) during smoking that initiate addiction. Nicotine-induced upregulation is the long-lasting increase in nAChR radio-ligand binding sites in brain resulting from exposure. The mechanisms causing upregulation are not established. Many different mechanisms have been reported with the assumption that there is a single, underlying cause. Using live cortical neurons, we examined for the first time how exposure and withdrawal of nicotine shape the kinetics of native α4β2-containing nAChR upregulation in real time. Upregulation kinetics demonstrate that at least two different mechanisms underlie this phenomenon. First, a transient upregulation occurs that rapidly reverses, faster than nAChR degradation, and corresponds to nAChR conformational changes as assayed by conformational-dependent, subunit-specific antibodies. Second, a long-lasting process occurs correlating with increases in nAChR numbers caused by decreased proteasomal subunit degradation. Previous radio-ligand binding measurements to brain tissue have measured the second process and largely missed the first. We conclude that nicotine-induced upregulation is composed of multiple processes occurring at different rates with different underlying causes.


Nicotine is the major addiction causing component in tobacco. Addiction is initiated by nicotine binding to high-affinity sites on nicotinic acetylcholine receptors (nAChRs) in brain. nAChRs are membrane proteins belonging to the cys-loop family of neurotransmitter-gated ion channels (Karlin and Akabas, 1995; Albuquerque et al., 2009). Neuronal nAChRs are comprised of eight different α subunits,α2–α7, α9–α10 and three other β subunits, β2–β4. Different nAChR subtypes are pentamers composed of two or more different α and β subunits, with the exception of the α-bungarotoxin binding subtype, which only contains α7 subunits (Drisdel and Green, 2000), resulting in distinct pharmacology and function in vivo (Sargent, 1993; McGehee and Role, 1995). The majority of high-affinity nicotine-binding sites in brain contain α4 and β2 subunits (Whiting and Lindstrom, 1988). In addition, there are other less characterized subtypes including receptors composed of α4, β2, and α5 subunits (Conroy and Berg, 1998), α3, β2 and β4 subunits (Xu et al., 1999; Parker et al., 2004) and α6 with other subunits (Klink et al., 2001; Cui et al., 2003; Parker et al., 2004).

Nicotine-induced upregulation is linked to different processes in nicotine addiction including sensitization (Vezina et al., 2007; Govind et al., 2009) and withdrawal (De Biasi and Dani, 2011). Upregulation occurs when nicotine exposure increases high-affinity nicotine binding sites in brain, measured by radio-labeled agonists such as nicotine (Marks et al., 1983; Schwartz and Kellar, 1983; Benwell et al., 1988; Breese et al., 1997) or epibatidine (Perry et al., 1999). Several factors limit studies examining the mechanisms underlying upregulation. First, the number of brain nAChR subtypes is not known because of their low levels. Another limitation is that radio-ligand binding to brain nAChRs is performed on membrane preparations or autoradiography sections and events that occur in live neurons may be missed. An alternative approach has been heterologous expression of different subunit combinations in mammalian cell lines or Xenopus oocytes (Peng et al., 1994; Hsu et al., 1996; Fenster et al., 1999). The disadvantage of heterologous expression is that nAChR subunit composition, while defined, may not correspond to that of native nAChRs. Non-neuronal cells may also lack brain-specific factors that regulate upregulation in brain.

Here, we assayed nicotine-induced upregulation of native nAChRs using live cortical neurons, which allowed real-time measurements of nAChR upregulation. We examined the initiation of upregulation and its reversal with nicotine withdrawal. The kinetics of native nAChR upregulation are biphasic revealing different processes that cause nicotine-induced upregulation. The first process is rapid and did not correlate with changes in the number of nAChRs, but did correlate with conformational-dependent binding of antibodies. The data are consistent with nAChR conformational changes causing transitions between a resting, low-affinity state and an upregulated, high-affinity state as previously proposed (Vallejo et al., 2005). The second process is much slower and correlated with slowed β2 subunit endoplasmic reticulum degradation, which causes increased subunit assembly and increased insertion of cell-surface nAChRs (Darsow et al., 2005). Nicotine-induced upregulation is, therefore, caused by multiple processes contrary to the assumption that upregulation results from a single underlying cause.

Materials and Methods

Antibodies used

Nicotinic acetylcholine receptor subunit specific antibody mAb299 (α4) was purchased from Covance (Princeton, NJ), mAb270 (β2) from DSHB at the University of Iowa, Anti-β2 SC (C-20) from Santa Cruz (Santa Cruz, CA), 6963 (α4), 4860 (α3) and 4886 (β4) were kind gifts from Prof. Scott Rogers, University of Utah, Salt Lake, UT, Anti-HA (Rabbit polyclonal) from Bethyl laboratories (Montgomery, TX), Anti-actin from Sigma-Aldrich (St. Louis, MO) and Anti-Glutamate decarboxylase (GAD) 65 from Millipore (Billerica, MA). Anti-Glial Fibrillary Acidic Protein (GFAP) and Anti-Microtubule Associated Protein (MAP-2) were both purchased from Cell Signaling Technology (Danvers, MA). Secondary antibody anti-Rabbit -HRP was obtained from Chappel, Anti-Goat -HRP from Santa Cruz (Santa Cruz, CA), Alexa Fluor anti-rat 488/568, Alexa Fluor anti-rabbit 647 and Alexa Fluor anti-Mouse 568 were purchased from Molecular probes (Eugene, OR).

Cell culture

HEK 293 cells (tSA201) stably expressing α4β2 nicotinic acetylcholine receptors were generated in our laboratory and the β2 subunit expresses an HA epitope at the C-terminus as previously described in (Vallejo et al., 2005). Cells were maintained in DMEM supplemented with 10,000U/ml pencillin and streptomycin (Invitrogen, Carlsbad, CA, MD), 10% calf serum (Hyclone, Logan, UT) and Hygromycin B (EMD chemicals, Gibbstown, NJ) at 37°C in the presence of 5% CO2.

Primary cultures of rat cortical neurons

Cortical neurons were cultured using protocol listed under Johns Hopkins Medicine website (http://www.hopkinsmedicine.org/institute_basic_biomedical_sciences/research_centers/metabolism_obesity_research/protocols/) with minor modifications. Cortex from Sprague Dawley rat pups at embryonic day 18 (E18) were dissected out and cut into small pieces. To dissociate the cells, the cortical pieces were incubated in 0.25% trypsin (Invitrogen, Carlsbad, CA) for 30 minutes, followed by washing once with Fetal Bovine Serum (Hyclone, Logan, UT) and thrice with Hanks balanced salt solution (Invitrogen, Carlsbad, CA). After the final wash the tissue was titurated using fire polished glass pipets. The dissociated cells were passed through a 70 micron cell strainer (BD Biosciences, San Jose, CA) to remove debri. The cells were centrifuged at 1500 rpm for 5minutes and re-suspended in neurobasal medium supplemented with B27 (Invitrogen, Carlsbad, CA). The cells are plated in 6cm tissue culture dishes pre-coated with poly-D-Lysine (Sigma-Aldrich, St Louis, MO) with an average density of 2 × 106. Half the media was replaced every three-four days until the experiments were done.

Metabolic labeling, immunoprecipitation and affinity purification

Newly synthesized subunits were labeled with 35S Methionine and Cysteine (1175 Ci/mmol; Easy tag EXPRESS S35 protein labeling mix, Perkin Elmer Life and Analytical Science, Shelton, CT). Cultures were starved in methionine-free DMEM for 15 minutes at 37°C, incubated in 5% CO2 and labeled in 1.5ml of methionine free medium supplemented with 111uCi of the 35S mix for 30 minutes. After labeling cells were washed thrice with ice cold DMEM and maintained in DMEM at 37°C, incubated in 5% CO2 in the presence or absence of nicotine for varying time intervals. At each chase time point, cells were scraped off from the plate in 1 ml PBS, centrifuged at 2400 rpm for 3 minutes, resuspended in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02% NaN3, plus 1% Triton X-100) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide, and chymostatin, pepstatin, leupeptin, and tosyllysine chloromethyl ketone at 10 μg/ml). Insoluble material was pelleted by centrifugation and resultant 35S-labeled lysates were incubated with anti-HA polyclonal antibody, mAB270 (β2), AChR β2 C-20 (β2), mAb 299 (α4) or 6963 (α4) for overnight at 4°C. Antibody-antigen complexes were precipitated with protein G-Sepharose (Amersham Biosciences, Arlington Heights, IL), and purified subunits were analyzed on a 4–8% gradient -Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was dried and exposed to bio-rad screen and the auto-rad was developed using a bio-rad molecular imager pharos-FX (Biorad, Hercules, CA). Quantification was done using Image J.

Nicotine induced upregulation and 125I-epi binding

HEK cells stably expressing α4β2HA maintained at 37°C in DMEM were treated with 10 μM nicotine for 17 hours. For intact counts, cells were washed three times with PBS and collected by gentle agitation with PBS followed by incubation with 5 nM 125I-epi (2200 Ci/mmol, Perkin Elmer Life and Analytical Science, Shelton, CT) for 20 minutes. The cells were harvested onto filter paper GF/B filters presoaked in 0.5% polyethyleneimine and washed with PBS using a 24 channel cell harvester (Brandel, Gaithersburg, MD). Radioactivity of bound 125I-epi was determined by γ-counting (Perkin Elmer- Wallac, Norton, OH). Non-specific binding was estimated by 125I-epi binding done on parallel samples pre-incubated with 1 mM nicotine.

To obtain antibody precipitated counts, the cells were solubilized in lysis buffer (see above) following 17 hours of nicotine exposure. Insoluble material was pelleted by centrifugation which removed ~25% of the available 125I-epi binding sites. The lysates were subjected to immunoprecipitation with various subunit specific antibodies. The antigen-antibody complexes were pulled down using protein G-Sepharose beads (Amersham Biosciences, Arlington Heights, IL). Following immunoprecipitation the beads were bound with 2.5 nM 125I-epi for 1 hour. Binding was done in the presence of 1 mM nicotine to determine the non-specific counts. The radioactivity associated with the beads was determined using γ-counting. The antibodies used were tested on cell lines expressing α4β4, α3β2 or α6β2 to determine their specificity. The β2 specific antibodies did not precipitated measurable 125I-epi counts from α4β4 expressing cells and α4 specific antibodies did not precipitate 125I-epi counts from α3β2 or α6β2 expressing cells.

Primary cultures of cortical neurons were treated with 1 μM nicotine for 17 hours. The cells were gently scraped off the plates and resuspended in 1nM 125I-epi for 20 minutes. The neurons were vacuum harvested to GF/B filter (Brandel, Gaithersburg, MD) and the specific radioactivity associated with the filtres was measured as described above. In the case of antibody precipitation, the neurons were exposed to nicotine as described above and lysed with lysis buffer containing 1% triton-X-100 and protease inhibitors. The neuronal lysates were incubated with subunit specific antibodies overnight followed by protein G-Sepharose precipitation. The immuprecipitated receptors were bound with 0.5 nM 125I-epi for 1 hour. The beads were washed three times with lysis buffer and the radioactivity remaining on the beads were determined using γ-counting.

RT PCR analysis

Total RNA was is olated from primary cortical cultures using Purelink RNA mini kit, Invitrogen (Invitrogen, Carlsbad, CA). Total RNA was reverse transcribed using random hexamer primers and multiscribe reverse transcriptase according to manufacturer’s protocol (Applied Biosystem’s RT kit, Foster city, CA). Reactions lacking RNA or reverse transcriptase served as control for DNA contamination or genomic amplification respectively. PCR primers were designed based on published protocols (Sheffield et al., 2000; Rogers et al., 2001) and the conditions used therein were followed. Primer pairs are listed for each nicotinic receptor along with the size of the PCR product in parenthesis: nAChRβ2, sense 59-GGAGTGGGAAGATTACCGCCTCA, antisense 5′-AGTCGTCGTGGTTCTCGTTGCG; nAChRβ3 sense 5′-GCTGAACACGAAGACGC, antisense 5′-GCAAAGACAGTCACC; nAChRβ4, sense 5′-AGAGTGCCTGCAAGATTGAG, antisense 5′-AGCTGACTGCAGACTTAGGA; nAChRα2 sense 5′-GATCTGGATCCCAGACATTG, antisense 5′-CGCCGATGAGTGGGATGACC; nAChRα3, sense 5′-GGTGGATGACAAGACCAAAGC, antisense 5′-AGGGCAGGTAGAAGACAAGCA; nAChRα4, sense 5′-CACGGTCTTCGTGCTCAATGT, antisense 5′-CCTTGGTTGCAGATGTCACTC; nAChRα5, sense 5′-GCTGCGCTGCTCTTGATGGT, antisense 5′-CGTATGTCCACGAGCCGAAT; nAChRα6 sense 5′ GCTTCATCCGGCCAGTGG, antisense 5′ GCAAAGAGTCACTTTCTCG; nAChRα7, sense 5′-CGCTGGTTCCCTTTTGATGTG, antisense 5′-CTATCAAGGGCACAGAATCAG; β-actin, sense 5′-AAGATCCTGACCGAGCGTGG, antisense 5′-CAGCAGTGTGTTGGCATAGAGG; ChAT sense 5′-GCCACTTGCATAGGTGAGGGC, Antisense 5′-GCCACTAGTCAGTTGGGC.


Cortical neurons were plated on coverslips pre-coated with Poly-D-Lysine. On 11 days in vitro (11DIV), neurons were fixed with 4% paraformaldehyde for 10minutes, quenched with 100 mM glycine for 10 minutes and permeabilized with 0.1% Triton X 100 for 10minutes. Blocking of nonspecific sites was performed with 5% normal donkey serum and 0.1% fish gelatin in PBS for 1 h before incubating with primary antibodies in blocking solution for 1hour. Antibodies and dilutions used were as follows: MAP-2 (1:1000), GFAP (1:1000), GAD-65 (1:1000), mAb299 (1:500) and mAb270 (1:500). Cells were then incubated in appropriate secondary antibodies (1:1000) for 1 h at room temperature. In some cases, nuclei were labeled with Hoechst dye for 5 min. Cells were mounted in ProLong gold and allowed to cure for 24 h before imaging on the Olympus DSU spinning disk (Integrated Microscopy Facility, University of Chicago). Samples prepared without primary antibody served as control for background fluorescence.

Biotinylation assays

Cell surface proteins were biotinylated using 0.5 mg/ml EZ-link sulfo-NHS-SS-Biotin (Thermo fisher scientific, Ashville, NC) for 20 minutes on ice. The cells were washed three times with PBS followed by DMEM and maintained in DMEM for the remaining time of chase. At the end of the chase period, cells were washed two times with PBS and solubilized in lysis buffer containing 1% triton-x-100 and protease inhibitors. Lysates were incubated overnight with streptavidin agarose (EMD chemicals Inc, Gibbstown, NJ). The biotinylated receptors were detected by either 125I-epi binding to beads or by immunoblots using subunit specific antibodies. In the instance of 125I-epi binding, the streptavidin agarose beads were incubated with 2.5 nM 125I-epi for 1 hour. To determine non-specific binding 125I-epi binding was done in the presence of 1mM nicotine. The beads were spun down by centrifugation at high speed for 30 seconds and washed three times with lysis buffer. The radioactivity remaining on the beads were determined using γ-counting. (Perkin Elmer-Wallac, Norton, OH). When subunits where detected by immunoblots, the biotinylated proteins were eluted from streptavidin agarose beads using 2x concentration of SDS sample buffer, denatured by boiling and subjected to SDS PAGE. The proteins were electro-transferred to PVDF membrane (Bio-Rad, Hercules, CA) and blots were probed with antibodies specific for α4 (6963) and β2HA (anti-HA).

Proteasome inhibitors

Cells stably expressing α4β2HA were treated with the proteasomal inhibitors 1 μM MG132, 1 μM lactacystin, or 10 μM nicotine for 4 hours. At the end of the incubation, cells were scraped and resuspended in PBS and subjected to 125I-epi binding. A parallel set of samples treated with nicotine or MG132 were lysed in lysis buffer (150mM NaCl, 5mM EDTA, 50mM Tris, pH 7.4, 0.02% NaN3, plus 1% Triton-X-100) containing protease inhibitors (2mM phenylmethylsulfonyl fluoride, 2mM N-ethylmaleimide and chymostatin, pepstatin, leupeptin, and tosyllysine chloromethyl ketone at 10ug/ml) and whole cell lysates were subjected to SDS PAGE analysis, proteins were transferred to PVDF membranes, and western blots were probed with 6963 (α4), anti-HA (β2HA) or anti-actin (loading control).

Primary cultures of cortical neurons (10DIV) were treated with proteasomal inhibitors 1 μM MG132, 1 μM lactacystin, or 1 μM nicotine for 17 hours and 125I-epi binding was done on intact neurons. Parallel samples treated with nicotine and MG132 were processed as described above and subjected to SDS-PAGE. Proteins were transferred to PVDF membranes followed by western blottting using antibodies 6963 (α4) and anti-actin. Alexa Fluor anti-Rabbit 647 was used to detect the α4 subunits and actin. The blots were scanned using Biorad Pharos-FX laser scanner. The band intensities were quantified using Image J software. (NIH).

Statistical analysis

Results are expressed as mean ± SEM of n samples. Statistical significance was assessed by a two-tailed Student’s t test.


nAChR high-affinity binding sites in live cortical neurons

Using cultured rat cortical neurons, we measured high-affinity 125I-epibatidine (125I-epi) binding to endogenous nAChRs. Neurons, 10 to 12 days in vitro (DIV), were exposed to 1 μM Nicotine for 17 hours to examine the increase or “upregulation” in 125I-epi binding to live neurons. Nicotine treatment caused a 3-fold increase in 125I-epi binding on average (27.6 ± 2.4 SEM fmole/mg total protein for untreated, 70.4 ± 4.5 SEM fmole/mg total protein for nicotine treated) and varied over a range of 2 to 4-fold. By varying the concentration of nicotine in the medium, we assayed the nicotine dose-dependence of the upregulation of the 125I-epi binding for cortical neurons (Figure 1A). An EC50 value of 92 nM was obtained by fitting the Hill equation to the data. Using a number of different techniques, we examined the subunit composition of native nAChRs in cortical neurons. Reverse transcription PCR was used to establish which mRNA transcripts were expressed in our cultures (Figure 1B). We detected α3, α4, α7, β2 and β4 subunit transcripts but not α2, α5, α6 and β3 subunit transcripts with oligonucleotide sequences already established to detect nAChR subunit transcripts (Sheffield et al., 2000; Rogers et al., 2001). α7 subunits primarily assemble into homomeric nAChRs that bind 125I-α-Bungarotoxin, but not radio-labeled agonists such as 125I-epi (Clarke et al., 1985; Harfstrand et al., 1988; Pauly et al., 1989). We, therefore, assayed α3, α4, β2 and β4 subunits at the protein level using subunit-specific antibodies (Abs) for each subunit. Abs specific for each of these subunits were used to precipitate nAChRs from the lysate and 125I epi binding performed on the immunoprecipitated nAChRs. The percentage of available epibatidine binding sites precipitated by each of the subunit-specific antibodies was determined. α4- and β2-specific Abs precipitated a significant percent of the available 125I-epi binding sites in the cortical neurons while relatively few of the 125I-epi binding sites were precipitated by α3- and β4-specific Abs (Figure 1C). Similarly using immunoblots, α4 subunits were immunoprecipitated by α4- and β2-specific Abs (Figure 1D). The expression of native “α4β2” receptors was further assayed using immunofluorescence microscopy (Figure 1E and F). α4- and β2-specific Abs only stained neurons that were labeled with MAP2 Abs and not astrocytes labeled with GFAP Abs (Figure 1E). α4-specific Abs stained inhibitory neurons labeled with GAD65 Abs and other neurons presumed to be excitatory (Figure 1F). Altogether, our results indicate that the native high-affinity 125I-epi binding sites that are upregulated by nicotine in the cortical neurons contain primarily α4 and β2 subunits and are the “α4β2” subtype.

Figure 1
nAChR nicotine-induced upregulation in cortical neurons

Real time kinetics of nicotine-induced upregulation onset and reversal

We next measured how exposure of cortical cultures to nicotine altered high-affinity 125I-epi binding to native nAChRs in real time. Cortical neuronal cultures were treated with 1 μM nicotine for varying times and 125I-epi binding were determined for nicotine-treated and untreated cultures at the different time points. The ratio of 125I-epi binding of nicotine-treated to untreated cultures or the fold-increase in 125I-epi binding was used as a quantitative measure of nicotine-induced upregulation. The time course of upregulation after application of nicotine is displayed in Figure 2A. The onset of upregulation occurred at two different rates. First, there was a fast component that saturated four hours after exposure and later a slower component that increased subsequently with continued nicotine exposure. Consistent with the presence of two separate processes, the data were only well fit as the sum of two exponential functions with a fast component (tau value (τ) =1.1 hours) and a slower component (τ = 12.5 hours).

Figure 2
Upregulation onset and reversal of cortical neuron nAChRs in real time

The two components of nicotine-induced upregulation were more evident when nicotine was removed from the medium after both components of upregulation were saturated with nicotine treatments (Figure 2B). Nicotine withdrawal initially caused a rapid decrease in the 125I-epi binding fold-increase. This fast component was followed by a much slower decay that continued days after nicotine withdrawal (see insert, Figure, 2B). The decay rate following nicotine withdrawal was again well fit as the sum of two separate processes. The fast component decayed with a τ value of 0.45 hours and the slow component decayed with a τ value of 12.3 days. The slow component of the decay of nicotine-induced upregulation after nicotine removal was not observed if cultures were exposed to nicotine 4 hours or less (Figure 2C). The kinetics of the fast component of upregulation during onset were similar to the fast decay during withdrawal and the slow onset of upregulation were similar to the slow component of decay. These results suggest that the fast components of upregulation represent the same process either turning on during onset or turning off during reversal. They also suggest that the slow components are a second, slow process turning on and off. In summary, we find that the nicotine-induced upregulation kinetics contain two components indicating that at least two different processes are causing upregulation. We also observed a similar biphasic decay of nicotine-induced upregulation for α4β2 and α6β2 receptors heterologously expressed in HEK293 cells (Figures 2D and E).

The kinetics of nAChR degradation during nicotine withdrawal

We began to test what mechanisms underlie the two different rates at which decay of upregulation occurred. A previous study from our laboratory found that changes in high-affinity binding with nicotine-induced upregulation did not correlate with changes in the number of α4β2 nAChRs stably expressed in HEK293 cells (Vallejo et al., 2005). However, if 125I-epi binding is a measure of receptor numbers, then the rates of decay of upregulation with nicotine withdrawal should correlate with the rate of degradation of the mature receptor complexes that bind 125I-epi. To determine the rates of nAChR subunit assembly and degradation, we used a pulse-chase approach to label the subunits with radio-labeled amino acids (e.g. (Merlie and Lindstrom, 1983; Green and Claudio, 1993; Sallette et al., 2005). Unfortunately, these experiments could not be performed using cultured cortical neurons because of the limited number of neuronal cells and relatively low levels of nAChR expression. As an alternative, we performed pulse-chase measurements on rat α4 and β2 subunits stably expressed in HEK cells that were previously used to study nicotine-induced upregulation (Vallejo et al., 2005), which also displayed fast and slow phases of the decay of upregulation following nicotine withdrawal (Figure 2D). The subunits in the α4β2 stable cell line were metabolically labeled with 35S-methionine/cysteine for 15 minutes. Subsequently, cells were solubilized at various chase times following nicotine withdrawal. Subunits were immunoprecipitated with several different α4- and β2-specific Abs 7–9 hours following nicotine removal and intensities of 35S-labeled bands measured. Examples of the results are displayed in Figure 3A. Our results were similar to previous pulse-chase analysis performed on human α4 and β2 subunits transiently expressed in HEK cells (Sallette et al., 2005). Subunit assembly as assayed by increases in subunit co-precipitation peaked over the first 60–90 minutes. The peak in subunit assembly was followed by a period over the next 5–8 hours where subunit degradation predominated as assayed by decreases in subunit band intensities. Differences in the assembly of the subunits were observed depending on which Ab was used and these differences are further described in more detail later (Figure 6). However, the rate of subunit degradation was independent of the Ab used for immunoprecipitation.

Figure 3
α4 and β2 subunit degradation after nicotine withdrawal
Figure 6
Nicotine alters binding of nAChR conformation-dependent Abs

The averaged results of the 35S-labelled band intensities precipitated by the different subunit-specific Abs are displayed in Figures 3B and C and were used to approximate rates of degradation of the α4 and β2 subunits after nicotine withdrawal. The rates of degradation of the newly synthesized subunits were relatively fast (τ values 3–6 hrs) but significantly slower than the fast phase of the decay of upregulation after nicotine withdrawal (τ value of 0.76 hrs; see Table 1). The degradation rate was not significantly different whether the cells were treated or untreated with nicotine. As discussed by Sallette et al. 2005, the kinetics of α4 and β2 subunit assembly and degradation in the ER are complex and are the combination of several processes including the degradation of unassembled subunits, the assembly of immature subunit complexes, and the maturation of the subunit complexes. Surprisingly, the degradation rate for the newly assembled subunits (subunits that co-precipitated) was not significantly different from the degradation rate for total cellular pool of subunits or the rate for unassembled subunits determined by stable expression of α4 subunits alone (data not shown). Using the pulse-chase analysis, we primarily precipitated a rapidly degrading pool of labeled subunits. The pool of mature nAChRs that is trafficked to the cell surface and degraded at a slower rate is a small proportion of the total subunit pool that is labeled during synthesis. Following loss of the rapidly degrading pool of labeled subunits, the levels of remaining subunits indicates that less than 20–30% of newly synthesized subunits are further assembled into the mature pool of nAChRs (Figures 3B and C).

Table 1
Parameters for the nicotine-dose dependence (EC50 values) and kinetics (τ values) for upregulation (onset and reversal) of native and heterologously expressed nAChR subtypes obtained from the relevant equation fit to the data (see Figure 1B, ...

Next we used cell surface biotinylation to assay the degradation rate of mature cell surface nAChRs after nicotine withdrawal (Figure 3D). Cell impermeable biotinylation reagents were used to biotinylate nAChRs on the surface of intact cells. Surface nAChRs on intact cells, were briefly biotinylated with membrane impermeable reagents at the end of the nicotine treatment (10 μM nicotine, 17 hours). After biotinylation and nicotine treatment, the cells were chased for the indicated times in the absence of nicotine and subunit band intensities were determined from immunoblots of the surface biotinylated subunits (Figure 3D). The decay of the surface receptor α4 and β2 subunit bands as a function of time after biotinylation and nicotine withdrawal in Figure 3D is a measure of the rate of degradation of the surface nAChRs. We observed no significant differences in the rates of degradation for the α4 and β2 subunits whether the surface nAChRs had been nicotine-pretreated or untreated. The decay of the band intensites after nicotine withdrawal was well fit as a single exponential process with a mean τ of 35 hours. Thus, mature α4β2 nAChRs are degraded at rates much slower than the newly assembled receptor subunits assayed in Figures, 3B and C, consistent with the degradation rates that had been previously measured for α4β2 nAChRs (Darsow et al., 2005) and other nAChRs (Christianson and Green, 2004).

We also examined the kinetics of 125I-epi binding to the plasma membrane pool of α4β2 receptors after nicotine withdrawal. To do this, 125I-epi binding was performed on the solubilized, surface biotinylated α4β2 nAChRs as a function of time after nicotine withdrawal (Figure 3E). The reversal of 125I-epi binding to the surface biotinylated nAChRs displayed both the fast and slow components similar to that observed when the 125I-epi binding was done on the whole cell population of nAChRs (Figure 2B and 2D). Because the fast component was not observed when the degradation of surface nAChRs was assayed (Figure 3D), the 125I-epi binding that rapidly reverses after nicotine withdrawal in Figure 3E cannot be caused by surface nAChR degradation or any change in surface nAChR numbers. These findings regarding the rapid phase of nicotine-induced upregulation are in agreement with our previous conclusions about mechanisms underlying nicotine-induced upregulation for α4β2 nAChRs stably expressed in HEK293 cells (Vallejo et al., 2005). However, in the previous study, we did not measure the effects of nicotine withdrawal and, thus, did not observe the second component of upregulation. We find that the slow decay rate of the second upregulation component (τ-45 hours, see Table 1), unlike the first, does correlate with the rate of degradation of the surface nAChRs (mean τ–35 hours). These results demonstrate that a second set of mechanisms, which change the number of nAChRs, cause the slow component of nicotine-induced upregulation.

Kinetics of degradation of nAChRs during nicotine exposure

During nicotine withdrawal, the degradation rate of newly synthesized subunits did not differ between cells that were nicotine pre-treated or left untreated (Figures 3A, B and C). However when nicotine-treatment was maintained (10 μM nicotine for 17 hours), we observed significant differences in the degradation rate for newly synthesized β2 subunits in the total and assembled pool, but less so for α4 subunits (Figure 4A, B and C). The degradation rate of mature nAChRs on the cell surface was not altered by nicotine treatment as shown previously (Darsow et al., 2005; Vallejo et al., 2005). Thus, maintained exposure to nicotine slows the degradation rate of newly synthesized subunits, largely β2 subunits (Figure 4B) leading to increased assembly of receptors.

Figure 4
α4 and β2 subunit degradation during nicotine exposure

Proteasome-mediated degradation of nAChR subunits causes the slow component of upregulation

We next examined the mechanisms causing the slow phase of nicotine-induced upregulation. Degradation of the newly synthesized subunits is likely occurring in the ER, similar to other nAChRs (Christianson and Green, 2004; Grailhe et al., 2004). We had found that ER-associated degradation (ERAD) of nAChR subunit was mediated by proteasomes as evidenced by increases in subunit levels and their assembly in the ER after blocking proteasome activity (Christianson and Green, 2004). To similarly test whether a loss of proteasome activity results in increases in α4 and β2 subunit levels in the ER we compared the effects of the proteasome inhibitor, MG132, and nicotine treatment on the levels of α4 and β2 in the α4β2 expressing HEK cell line. Treatment of the cells with MG132 caused a 2-fold increase in the steady-state levels of both α4 and β2 subunits (Figure 5A, 1st, 2nd and 3rd panel). Nicotine treatment resulted in a 2-fold increase in β2 subunit steady-state levels (Figure 5A 1st and 3rd panel) but had a much smaller effect on α4 subunit levels (1.3-fold; Figure 5A, 1st and 2nd panel), consistent with the greater effect nicotine had on the rate of β2 subunit degradation compared to α4 subunit degradation (Figure 4B and C). In contrast to a previous study, which suggested that nicotine directly inhibits proteasomal activity (Rezvani et al., 2007), we observe a differential effect of nicotine treatment on α4 and β2 subunit levels compared to the proteasomal block suggesting that nicotine is not directly inhibiting proteasomal activity in the conditions measured in this study. We obtained additional evidence that nicotine is not directly inhibiting proteasomes when α4 and β2 subunits were stably expressed individually in HEK cells (Figure 5B). Proteasomal inhibition still increased α4 and β2 subunit levels 2-fold, but nicotine treatment had no effect onα4 and β2 subunit levels when expressed individually (Figure 5B, 1st, 2nd and 3rd panels). These results suggested that the nicotine-induced effects on α4 and β2 subunit levels and degradation rates require the presence of both subunits and subunit assembly in cells.

Figure 5
Nicotine slows proteasomal degradation of α4 and β2 subunit

Inhibition of proteasomes had similar effects on nicotine-induced upregulation of α4β2 nAChRs in cortical neurons. Only α4 subunit levels could be measured using immunoblots for cortical neurons. Similar to the α4β2 HEK cell line, proteasomal inhibition and nicotine-induced upregulation increased α4 subunit levels, a 1.7-fold increase with MG132 treatment and a 1.2-fold increase with nicotine treatment (Figure 5C, 2nd panel). A significantly smaller fold-increase in 125I-epi binding was observed with protease inhibitors (MG132 and lactacystin) than for nicotine-induced upregulation for both cortical neurons (Figure 5C, 3rd panel) and α4β2 expressing cells (Figure 5A, 4th panel) even though proteasomal inhibition increased subunit levels to a greater extent. Thus, it appears that nicotine binding to pre-existing nAChRs induces a decrease in proteasome-mediated degradation of nAChR subunits, primarily β2 subunits, during ERAD. Slowing of β2 subunit ERAD increases the number of subunits that assemble into nAChRs in the ER. Increased nAChR assembly, in turn, increases nAChR numbers and 125I-epi binding at intracellular and surface sites, similar to what was found previously with muscle nAChR subunits when ERAD was blocked (Christianson and Green, 2004).

Nicotine-induced nAChR conformational changes occur during the fast phase of upregulation

We also investigated the mechanisms responsible for the fast component of nicotine-induced upregulation that occur following nicotine withdrawal. In Figure 3, we found that the fast decay of nicotine-induced upregulation during nicotine withdrawal does not correspond to a change in the number of nAChRs. If not a change in the number of nAChRs, what causes the decay in 125I-epi binding? We had previously proposed that nicotine induces a conformational change during upregulation causing nAChRs to transition from a low-affinity, resting state to a high-affinity, upregulated state (Vallejo et al., 2005). Evidence of this conformational change during upregulation was a change in the α4β2 nAChR functional state with upregulation (Buisson and Bertrand, 2001; Vallejo et al., 2005). Another assay of nAChR conformational changes is the interaction between nAChRs and monoclonal Abs (mAbs) with conformational-dependent epitopes. Conformational-dependent mAbs have been used to assay for muscle-type nAChR subunit conformational changes that occur during nAChR assembly and maturation in the ER (e.g., (Merlie and Lindstrom, 1983; Green and Claudio, 1993).

We tested a number of α4- and β2-specific Abs to examine whether their associations with mature 125I-epi-bound α4β2 nAChRs were altered by nicotine treatment. To assay for changes in Ab-nAChR associations caused by nicotine treatment, we measured the percentage of 125I-epi binding that was precipitated by each Ab for nicotine-treated and untreated cells. This percentage was obtained from the ratio of Ab precipitated 125I-epi binding counts to total cellular 125I-epi binding for intact cells that were either nicotine treated or untreated. Using the stable α4β2-expressing cell line (Figure 6A), two of the Abs tested showed no change in the precipitation of 125I-epi-bound nAChRs with nicotine treatment. The polyclonal Abs tested precipitated a higher percentage of the total 125I-epi binding counts. Three of the Abs tested showed large increases in the precipitation of 125I-epi-bound nAChRs with nicotine treatment, but precipitated a smaller percentage of the total 125I-epi binding counts even for the nicotine treated cells. Two of the Abs that showed nicotine-induced changes in the precipitation of 125I-epi-bound nAChRs, β2-specific mAb 270 and α4-specific mAb 299, were mAbs whose interactions with nAChRs were previously shown to be dependent on subunit conformation (Harkness and Millar, 2002). These findings indicate that mAb 270 and 299 epitopes become more accessible for binding after nicotine treatment while the availability of the α4- and β2-specific polyclonal Ab epitopes does not change after nicotine treatment. Surprisingly, a third Ab, specific for the HA epitope that was fused to the β2 subunit C-terminus, also showed similar increases in the precipitation of 125I-epi-bound nAChRs with nicotine treatment suggesting that the HA epitope was changing with nicotine treatment becoming more accessible for Ab binding similar to mAbs 270 and 299.

We performed the same experiments with cortical neurons (Figures. 6C–D) and observed similar results. The HA-specific mAb could not be used because the native β2 subunits lack the HA epitope. The α4- and β2-specific polyclonal Abs precipitated the same percentage of 125I-epi bound nAChRs whether the neurons had been nicotine treated or untreated (Figure 6C and D). The β2-specific polyclonal Ab precipitated a smaller percentage of the 125I-epi bound nAChRs compared to the α4β2 cell line. The smaller percentage resulted from a new lot of β2 specific Ab that was less efficient for precipitation than the previous lot. The new lot also precipitated a similar, smaller percentage of 125I-epi bound nAChRs when repeated with the α4β2 cell line (data not shown). The α4-specific polyclonal Ab precipitated the same percentage of the 125I-epi bound nAChRs where the same lot of Ab was used. As with the α4β2 cell line, we observed a significant fold-increase in the percentage of precipitated 125I-epi binding for mAbs 270 and 299 with nicotine treatment. The fold-increase for, both mAbs was 2-fold, which is smaller than that observed in the α4β2 cell line, consistent with the smaller fold-increase of upregulation (Figure 6E). The percentage of the 125I-epi bound nAChRs precipitated by mAbs 270 and 299 was higher than the α4β2 cell line. The higher percentage precipitated by the conformation-dependent mAbs may be caused by a higher efficiency of mature nAChR formation in the cortical neurons relative to the stable HEK cell line.

In a recent study, 125I-epi binding measurements were performed on brain tissue from nicotine-or saline-treated mice. Contrary to what we observed for the native nAChRs in cortical neurons, nicotine treatment did not result in increases in mAb 270 or 299 associations with native nAChRs (Marks et al., 2011). The differences between their findings and ours could result from a two hour delay after nicotine treatment before sacrificing the mice brains for epibatidine binding. In our cortical cultures, we found that two hours after nicotine withdrawal, the fast component of upregulation had completely decayed and the fold-increase in 125I-epi binding changed from 2.9 to 1.7 (Figure 2B). Thus, the measurements made on brain tissue would have missed the fast component and would have only measured the slow component of nicotine-induced upregulation. Consistent with this, 125I-epi binding in the cortical regions was only increased 1.5-fold on average by the nicotine treatment when measured by immunoprecipitation and 1.7-fold by autoradiography. In a different study (Turner et al., 2011), both the fast and slow components of nicotine-induced upregulation were observed when mice were sacrificed without delay after a similar nicotine treatment and rapid membrane preparation. In this study, 3H-epi binding to the cortical regions was increased to significantly higher levels, 2.7-fold, by nicotine treatment similar to the levels we measured before nicotine was withdrawn from the cortical cultures. Furthermore, they found that the binding had decayed 24 hours after treatment to a level (1.6-fold) consistent with our measurements of the slow component and was maintained at that level for 3 days. The results of these two studies would be consistent with our data on cortical cultures if a nicotine-induced α4β2 conformational change, as assayed by the conformation-dependence of mAb precipitation of 125I-epi binding, is observed only during the rapid decay phase of the upregulation.

We, therefore, tested whether the nicotine-induced increases in mAb precipitations of nAChRs were only observed during the fast decay phase of the upregulation. For the cortical neurons, 5 hours after nicotine withdrawal is a time when the fast component had completely disappeared and the slow component had not changed. Hence 5 hours after nicotine withdrawal we used the same sets of antibodies to assay changes in the percentage of 125I-epi counts precipitated. At this time after nicotine withdrawal, the nicotine-dependent increases in the percentage of precipitated 125I-epi bound nAChRs had disappeared (Figure 6F), consistent with nicotine-dependent changes in epitope availability occurring as part of the fast component and not the slow component of upregulation. We also tested in α4β2 HEK cells whether conformational-dependent mAb precipitation of the 125I-epi bound nAChRs changed with the decay of the fast component of the upregulation (Figure 6E). At a time after nicotine withdrawal when the fast component has completely disappeared and the slow component had hardly changed (3 hours), the nicotine-dependent increases in the percentage of precipitated 125I-epi bound nAChRs had disappeared (Figure 6E). Thus for the stably expressed α4β2 nAChRs, as well as for the cortical neuron native α4β2 nAChRs, the nicotine-dependent increases in mAb precipitation only occur during the fast phases of the nicotine-induced upregulation.


While nicotine-induced upregulation of nAChRs is a well-established phenomena linked to nicotine addiction, its role in nicotine addiction and the mechanisms responsible for upregulation are not established. Nonetheless, many different mechanisms have been proposed to explain how upregulation occurs. These include mechanisms that increase nAChR numbers such as a decrease in nAChR cell surface turnover (Peng et al., 1994), increased nAChR trafficking to the surface (Darsow et al., 2005), increased nAChR subunit maturation and assembly in the ER (Harkness and Millar, 2002; Nashmi et al., 2003; Sallette et al., 2005) and block of nAChR subunit degradation in the ER (Ficklin et al., 2005; Rezvani et al., 2007). Additional mechanisms have been proposed to upregulate without increases in nAChR numbers such as a change in nAChR stoichiometry (Nelson et al., 2003; Moroni et al., 2006) and a nicotine-induced conformation change of nAChRs that alters nAChR ligand binding state and function (Vallejo et al., 2005). In these studies, a single mechanism was assumed to be the predominant cause of upregulation and the possibility of multiple mechanisms was not considered. Furthermore, all of the studies were performed on cells heterologously expressing nAChRs because of the difficulties involved in performing radio-labeled ligand binding measurements and other assays on brain tissue. As a consequence, how these studies relate to the native brain nAChRs in neurons is unclear.

In this study, we characterized the nicotine-induced upregulation of α4β2 nAChRs in cultured cortical neurons by measuring the kinetics of upregulation of native nAChRs in real time and in living brain-derived neurons. During long exposures of the neurons to nicotine (17–24 hours), 125I-epi binding increased at two rates with τ values of 1.1 and 12.5 hours. When nicotine was withdrawn, 125I-epi binding decayed at different rates, with τ values of 0.45 hours and 12.3 days. That the first process can occur independent of the second was demonstrated with shorter exposures of the cortical neurons to nicotine (4hours or less) where only the fast decay occurred (Figure 2C). During the initial process, nAChR numbers did not change and conformational-dependent binding of a set of nAChR-specific mAbs occurred (Figure 6D). Conformational-dependent mAb binding remained only during the rapid decay and was gone subsequently. In addition, 125I-epi binding to cell-surface nAChRs rapidly dissipated (Figure 3E) while the number of cell-surface nAChRs as assayed by immunoblot analysis remained essentially unchanged (Figure 3D). Based on these results, we conclude that the initial component of upregulation results from nicotine-induced nAChR conformational changes. We assume that the conformational changes alter 125I-epi binding because they reflect nAChR transitions from a “resting” state with negligible 125I-epi binding to an “upregulated state” with high-affinity 125I-epi binding as previously proposed (Vallejo et al., 2005).

We also characterized mechanisms responsible for the second component of upregulation. During the onset of this process in HEK cells, we observed increased nAChR assembly (Figure 4A) as previously observed (Sallette et al., 2005). A decreased rate of newly synthesized β2 subunit degradation (Figures 4A, B and C) accompanied nicotine-induced increases in nAChR assembly. Blocking proteasome-associated proteases increased subunit levels in cortical neurons and HEK cells (Figures 5A, 1stpanel and 5C, 1stpanel). The proteasome inhibition also increased 125I-epi binding in HEK cells and cortical neurons consistent with increases associated with the second component of the upregulation (Figures 5A, 4thpanel and 5C, 3rdpanel). In addition, the decay rate of the second process in HEK cells (τ-44 hours, Figure 2D) correlated with the rate of degradation of mature α4β2 nAChRs (mean τ-35 hours, Figure 3D). Altogether our results are consistent with a series of events starting with a mechanism protecting β2 subunits from degradation by proteasomes (e.g., (Rezvani et al., 2009)). The resultant increased levels of β2 subunits increases nAChR assembly in the ER and more intracellular and cell-surface nAChRs accumulate. The mechanisms causing the second component are, thus, distinct from those causing the first component of upregulation and result in significant changes in the number of upregulated α4β2 nAChRs.

Aspects of upregulation including its dose-dependence and its kinetics depended on the cell type (HEK cell vs. cortical neurons) and subunit composition (see Table 1). If the cortical neuron nAChR subunit composition is predominantly α4 and β2 as indicated by our data (Figures 1B and C), then other factors appear to cause the differences in α4β2 nAChR upregulation in the neurons from HEK cells. One can imagine that differences in posttranslational modifcations for example: phosphorylation (Bermudez and Moroni, 2006; Wecker et al., 2010), palmitoylation (Drisdel et al., 2004; Alexander et al., 2010), differences in interacting proteins (Jeanclos et al., 2001; Ficklin et al., 2005) or differences in the lipid environment (Fong and McNamee, 1986; Barrantes, 2010) could cause changes in how upregulation occurs. One striking difference between native α4β2 nAChRs and the α4β2 nAChRs expressed in HEK cells is the faster onset of upregulation and 3-fold lower EC50 value for the native subunits. These findings suggest that nicotine can initiate upregulation of native α4β2 nAChRs in a matter of minutes at nicotine concentrations lower than previously thought. However, short-term exposure to nicotine, such as those achieved during one or two cigarettes, would only trigger the first component of upregulation, which rapidly reverses.

How do the results of this study using cultured cortical neurons relate to upregulation in vivo? While nAChRs in the cortical neurons are predominantly α4β2 nAChRs, the situation is different in the reward areas of the midbrain where dopamine neurons contain multiple nAChR subtypes, in particular α6-containing nAChRs (Gotti et al., 2005; Salminen et al., 2007), which are likely to complicate detailed studies of upregulation. Some studies using brain tissue have measured the decay rates of upregulated radioligand binding at different times after nicotine withdrawal. Decay rates varied somewhat depending on how nicotine was administrated or which brain region was studied (Marks et al., 1985; Collins et al., 1990; Hulihan-Giblin et al., 1990; Collins et al., 1994). Yet, upregulated binding decayed slowly, over days, similar to what we observed for the second component of upregulation. As discussed above, animals were sacrificed two hour or longer after nicotine treatment ended, delaying the time between nicotine treatment and when radio-labeled agonist binding began. The delay allows nicotine metabolism to minimalize the levels of nicotine and its metabolites present. However, a consequence of the delay is that the fast component of upregulation would have been missed due to its decay prior to binding measurements. Evidence of this is a recent study where radio-labeled agonist binding was assayed without delay and the fast decaying component of the upregulated binding was observed (Turner et al., 2011). Therefore, with the exception of one study, radio-labeled agonist binding measurements in brain tissue have only measured the slow decaying component of upregulation.

Nicotine-induced upregulation has been assumed to be caused by a single process. Instead, we find here that upregulation is composed of multiple processes occurring at very different rates with different underlying causes. Our findings that upregulation consists of a series of different processes stimulated by nicotine raise the possibility that upregulation has a more extensive role in nicotine addiction than previously thought. With very different kinetics, multiple upregulation components may contribute to different features of nicotine addictive behavior including sensitization, withdrawal and tolerance, which occur at different times after nicotine exposure. This question would be ideally addressed if there existed a way to specifically block one component of upregulation without affecting other components. The specific blocker could then be applied to in an in vivo preparation where the behavioral features of nicotine exposure can be measured for changes that correlate with the block. Another question raised by these findings is whether the fast, initial component of upregulation is required for initiation of the slower second process or whether the second process is independent of the initial process. Indirect evidence that the two processes are linked is that the nicotine dose-dependence of both processes together is described by a single binding isotherm with a Hill slope of 1.8 and an EC50 value of 92 nM for the cortical neuron nAChRs (Figure 1A). If nicotine was acting through two independent processes, differences in the nicotine dose-dependence would be expected and we should have observed multiple components in the nicotine dose-dependence or at least a Hill slope of less than 1. Other nAChR subtypes with different subunit compositions are upregulated by nicotine with similar steep nicotine dose-dependences but with large differences in EC50 values for the nicotine dose-dependence (Wang et al., 1998; Fenster et al., 1999; Tumkosit et al., 2006; Walsh et al., 2008). One of these subtypes, α6β2 nAChRs (Figure 2E), exhibit biphasic kinetics of the decay of 125I-epi binding similar to the kinetics of the cortical neuron (Figure 2B) and stably expressed α4β2 nAChRs (Figure 2D). The upregulation of α6β2 nAChRs, like α4β2 nAChRs, thus, appears to consist of both components yet has greater than a 100-fold difference in the EC50 value of the nicotine dependence of upregulation (Table 1), These findings suggest that the multiple processes underlying nicotine-induced upregulation are initiated by nicotine binding to the same binding sites on nAChRs.


We thank Dr. Scott Rogers of generously providing some of the Abs used in this study. We also thank Dr. Okunola Jeyifous for helping with primary neuronal cultures of cortex and Dr. Sarah Antinone for critical reading and comments about this paper. This work was supported by grants: National Institutes of Health (NIH) NS043782, DA019695, the Peter F. McManus Foundation (WNG) and fellowships from the Marine Biological Laboratory in Woods Hole, MA.


Conflict of interest: None


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