Chronic Menthol Does Not Change Stoichiometry or Functional Plasma Membrane Levels of Mouse α3β4-Containing Nicotinic Acetylcholine Receptors

Abstract

Heteromeric α3β4 nicotinic acetylcholine (ACh) receptors (nAChRs) are pentameric ligand-gated cation channels that include at least two α3 and two β4 subunits. They have functions in peripheral tissue and peripheral and central nervous systems. We examined the effects of chronic treatment with menthol, a major flavor additive in tobacco cigarettes and electronic nicotine delivery systems, on mouse α3β4 nAChRs transiently transfected into neuroblastoma-2a cells. Chronic menthol treatment at 500 nM, near the estimated menthol concentration in the brain following cigarette smoking, altered neither the [ACh]-response relationship nor Zn²⁺ sensitivity of ACh-evoked currents, suggesting that menthol does not change α3β4 nAChR subunit stoichiometry. Chronic menthol treatment failed to change the current density (peak current amplitude/cell capacitance) of 100 μM ACh-evoked currents. Chronic menthol treatment accelerated desensitization of 100 and 200 μM ACh-evoked currents. Chronic nicotine treatment (250 μM) decreased ACh-induced currents, and we found no additional effect of including chronic menthol. These data contrast with previously reported, marked effects of chronic menthol on β2* nAChRs studied in the same expression system. Mechanistically, the data support the emerging interpretation that both chronic menthol and chronic nicotine act on nAChRs in the early exocytotic pathway, and that this pathway does not present a rate-limiting step to the export of α3β4 nAChRs; these nAChRs include endoplasmic reticulum (ER) export motifs but not ER retention motifs. Previous reports show that smoking mentholated cigarettes enhances tobacco addiction; but our results show that this effect is unlikely to arise via menthol actions on α3β4 nAChRs.

Introduction

Smoking is the leading cause of preventable death worldwide (https://www.cdc.gov/tobacco/data_statistics/fact_sheets/fast_facts/?index/html), and is responsible for ∼6 million deaths annually (https://apps.who.int/iris/bitstream/handle/10665/272694/9789241514170-eng.pdf?ua=1). Nicotine causes smoking addiction through binding to pentameric ligand-gated nicotinic acetylcholine receptors (nAChRs). Chronic exposure to nanomolar or micromolar concentrations causes upregulation of nAChRs at the plasma membrane (PM). This upregulation occurs partially because nicotine enters the endoplasmic reticulum (ER), binds there to nascent α4β2 nAChRs, acts as a post-translational pharmacological chaperone for these nAChRs, and consequently increases ER exit of these nAChRs via post-translational pharmacological chaperoning (Srinivasan et al., 2012b; Henderson and Lester, 2015). Nicotine upregulates nAChR α4 or β2 protein levels without changing their mRNA levels (Flores et al., 1992; Marks et al., 1992; Buisson and Bertrand, 2001; Srinivasan et al., 2011).

Compared with smokers of nonmentholated cigarettes, smokers of mentholated cigarettes have higher upregulation of α4β2-containing nAChR densities in the brain (Brody et al., 2013). There were reduced rates of smoking cessation among smokers of menthol-containing compared with nonmenthol-containing cigarettes at both 4-week and 6-month checkups (Gandhi et al., 2009).

There is a molecular explanation from mouse studies for the addictive properties of nanomolar-range menthol treatment (Henderson et al., 2016, 2017). Chronic treatment (∼24 hours) with menthol alone upregulates α4 and α6 nAChR subunits selectively in midbrain dopaminergic neurons in the ventral tegmental area and substantia nigra pars compacta (Henderson et al., 2016), whereas nicotine alone upregulates nAChR α4 number in GABAergic neurons of the substantia nigra pars reticulata (Nashmi and Lester, 2007; Xiao et al., 2009). Furthermore, chronic menthol treatment alone shifted stoichiometry toward lower sensitivity (α4)₃(β2)₂ and α6β2(non-β3) from higher sensitivity (α4)₂(β2)₃ and α6β2β3 nAChR populations, respectively (Henderson et al., 2016). While chronic menthol treatment alone prevented nicotine reward-related behavior (Henderson et al., 2016), combined chronic menthol and nicotine treatment further enhanced reward-related behavior caused by chronic nicotine alone (Henderson et al., 2017). Additionally, chronic menthol treatment enhances nicotine-induced upregulation of α4* and α4α6* nAChRs (Henderson et al., 2017).

The human α3-, α5-, and β4-nAChR subunit gene cluster located on chromosome locus 15q24–25.1 is linked to the risk of nicotine dependence and smoking-associated diseases, as well as to lung cancer among smokers (Saccone et al., 2007; Bierut et al., 2008; Spitz et al., 2008; Chen et al., 2009; Tobacco and Genetics Consortium, 2010). In a meta-analysis of 16 studies, 130 single nucleotide polymorphisms in 15q24–25.1 are associated with the number of cigarettes smoked a day, with rs1051730 in CHRNA3 having the strongest association (Tobacco and Genetics Consortium, 2010). Furthermore, subunits from the α3-, α5-, and β4-nAChR subunit gene cluster are expressed in the medial habenula-interpeduncular tract (Dineley-Miller and Patrick, 1992; Marks et al., 1992; Sheffield et al., 2000; Whiteaker et al., 2000, 2002; Grady et al., 2009; Shih et al., 2014) and contribute to nicotine dependence by influencing nicotine aversion in the medial habenula-interpeduncular midbrain pathway (Fowler et al., 2011; Frahm et al., 2011). Compared with patients who do not have lung cancer, α3 and β4 nAChR-encoding genes are overexpressed in small-cell lung carcinoma of lung cancer patients (Improgo et al., 2010). Agonist activation of α3β4 nAChRs can promote viability of these lung carcinoma cells, whereas antagonism or knockdown of α3β4 nAChRs reduces viability of these cells (Improgo et al., 2013).

Similar to the inhibitory effects at human α4β2 nAChRs (Hans et al., 2012), (−)-menthol, when coapplied with 30 µM acetylcholine (ACh), is a noncompetitive antagonist at human nAChR α3β4 (IC₅₀ = 100 µM), while also causing faster desensitization of ACh-evoked currents (Ton et al., 2015). It is of interest to observe chronic (∼24 hours) effects of a much lower, pharmacologically relevant concentration of menthol on α3β4 nAChRs, because the estimated concentration of menthol in a smoker’s brain is 0.5–2.5 µM (Henderson et al., 2016, 2017).

We examined effects of chronic menthol (500 nM, 24–30 hours) treatment alone and in combination with chronic nicotine on the two potential stoichiometries, (α3)₂(β4)₃ and (α3)₃(β4)₂, and on functional PM levels of mouse α3β4 nAChRs. The efficiency of assembly and trafficking of nAChR varies depending on the receptor subtypes and the cell system in which they are expressed (Crespi et al., 2018a). These nAChRs were transiently transfected in mouse neuroblastoma-2a (Neuro-2a) cells to determine whether α3β4 nAChRs mediate the addictive effects of menthol in mentholated cigarettes.

Materials and Methods

Reagents.

(−)-Menthol, (±)-menthol, (+)-menthol, (−)-nicotine hydrogen tartrate, and acetylcholine chloride were obtained from Sigma-Aldrich (St. Louis, MO). Menthol dose selection has been discussed previously and is based on an analysis estimating the concentration of menthol in the brain following a long-term exposure paradigm, as well as on preliminary concentration-response studies (Henderson et al., 2016, 2017).

Neuro-2a Cell Culture and Transient Transfection.

We used mouse Neuro-2a cells (American Type Culture Collection, Manassas, VA) for our experiments. Passage 3–20 Neuro-2a cells (50,000) were plated onto sterilized 12-mm-diameter glass coverslips (Deckgläser, Sondheim, Germany), which were placed in 35-mm culture dishes, and cultured in a humidified incubator (37°C; 95% air, 5% CO₂). Neuro-2a cells were incubated in full cell culture medium containing Eagle’s minimum essential medium, 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin.

For both Zn²⁺-inhibition experiments and [ACh]-response experiments (Figs. 1–3; Tables 1–3), 35-mm culture dishes containing preplated Neuro-2a cells were transfected with 125 ng of each nAChR subunit cDNA [mouse α3-green fluorescent protein (GFP) and mouse wild-type (WT) β4] in the pCDNA3.1 vector. These constructs were used in Shih et al. (2014) and contain a GFP tag within the M3-M4 loop of mouse α3 nAChR. Plasmids were mixed with 250 µl of Opti-MEM (Thermo Fisher Scientific Inc., Chino, CA) and Lipofectamine 2000 (Thermo Fisher Scientific Inc.) was separately mixed with 250 µl of Opti-MEM. After 5 minutes at 24°C, DNA- and Lipofectamine 2000–containing Opti-MEM solutions were mixed together and incubated for 25 minutes at 24°C. The solutions were then added to 35-mm culture dishes containing preplated Neuro-2a cells, which were then placed in the humidified incubator for 24 hours. The Opti-MEM was removed and replaced with full cell culture media containing either 500 nM (−)-menthol, 500 nM (+)-menthol, 500 nM (±)-menthol, 250 μM (−)-nicotine, combined 500 nM (−)-menthol and 250 μM (−)-nicotine, or neither menthol nor nicotine (control treatment) for 24–30 hours. Filter (0.2 μm)-sterilized menthol and nicotine stock solutions were used to make 500 nM menthol and/or 250 μM nicotine.

Fig. 1.

Functional characterization shows that chronic (24–30 hours) (−)-menthol and/or nicotine treatment does not change the Zn²⁺ sensitivity of ACh-evoked currents at mouse α3β4 nAChRs. cDNA-encoding mouse α3-GFP and WT β4 subunits were transfected at a 1:1 ratio into Neuro-2a cells. Using whole-cell patch clamp at a holding potential (−65 mV), inward current responses were recorded during ACh application at the indicated concentrations and the chronic treatment conditions were underlined. Menthol and/or nicotine were not present during ACh application. (A) [ACh]-response curves (average normalized response ± S.E. values are represented in the curve) were constructed (n = 6–13 for different concentrations). (B) Exemplar voltage-clamp current traces displayed by their ACh concentration and duration of application. (C) Chronic 500 nM (−)-menthol (n = 16), chronic 250 μM nicotine (n = 19), and combined chronic 500 nM (−)-menthol and 250 μM nicotine (n = 20) treatments did not significantly (n.s.) change the level of inhibition of 100 μM ACh by 1 mM Zn²⁺ compared with control (n = 14) treatment (P > 0.05 for both one-way ANOVA with post-hoc Tukey HSD test and two-tailed t tests). Mean ± S.E. values are represented in the bar chart. The 1 mM Zn²⁺ solution without ACh was applied to the Neuro-2a cells for 2 seconds before it was coapplied with 100 μM ACh for 1 second. (D) Exemplar traces of 100 μM ACh only application 2.5 minutes before, during coapplication, and 100 μM ACh only application 2.5 minutes after (washout) 100 μM ACh + 1 mM Zn²⁺ coapplication. The black bar represents ACh application for all three traces, and the adjoining gray bar represents the remaining ACh application for one to two traces (differences in application due to the variations in flow rates from the Octaflow manifold that combined solutions).

Fig. 3.

Chronic (24–30 hours) (−)-menthol treatment does not alter functional PM levels of mouse α3β4 nAChRs transiently transfected into Neuro-2a cells. Summary of current density (-pA/pF) for the different chronic treatments [control, 500 nM (−)-menthol, 250 μM nicotine, and combined 500 nM (−)-menthol and 250 μM nicotine; n = 27, 28, 19, and 20, respectively]. n.s., not significantly changed (P > 0.05) by one-way ANOVA with post-hoc Tukey HSD test. Mean ± S.E. values are represented in the bar chart. Menthol and/or nicotine were not present during ACh application.

TABLE 1.

Level (%) of 1 mM Zn²⁺ inhibition of 100 µM ACh currents at mouse α3β4 nAChRs under different chronic (24–30 h) treatments

Cells were studied 52.5–52.75 h after the start of transfection and 26.5–27.25 h after the start of incubation in no drug-containing media or after addition of menthol and/or nicotine (both durations are averages). Mean ± S.E. values are represented in the table.

Chronic Treatment	Control	(−)-Menthol	Nicotine	Nicotine and (−)-Menthol
(−)-Menthol (nM)	0	500	0	500
Nicotine (µM)	0	0	250	250
% Zn2+ inhibitiona (%)	50.1 ± 6.5	48.5 ± 4.8	48.7 ± 6.1	55.4 ± 5.3
N	14	16	19	20
Cell capacitance (pF)a	28.2 ± 5.3	23.3 ± 2.5	22.5 ± 1.9	22.9 ± 1.9
Current density (pA/pF)a,b	−108.4 ± 31.6	−91.4 ± 15.6	−62.7 ± 10.4	−71.9 ± 12.3

^aP > 0.05 for one-way ANOVA with post-hoc Tukey HSD test.

^bCurrent density between these same chronic treatments with higher N numbers for control and chronic (−)-menthol treatment are shown in Table 3 and discussed in Results in the section entitled Chronic Nicotine But Not Chronic Menthol Treatment Reduces Functional PM Mouse α3β4 nAChR Levels.

TABLE 3.

Current density (pA/pF) at mouse α3β4 nAChRs under different chronic (24–30 h) treatments

Cells were studied 52.5–52.75 h after the start of transfection and 26.5–27.0 h after the start of incubation in no drug-containing media or after addition of menthol and/or nicotine (both durations are averages). Mean ± S.E. values are represented in the table.

Chronic Treatment	Control	(−)-Menthol	Nicotine	Nicotine and (−)-Menthol
(−)-Menthol (nM)	0	500	0	500
Nicotine (µM)	0	0	250	250
Cell capacitance (pF)a	24.1 ± 3.0	21.9 ± 2.1	22.5 ± 1.9	22.9 ± 1.9
Current density (pF/pA)a	−129.7 ± 22.9	−102.7 ± 19.6	−62.7 ± 10.4	−71.9 ± 12.3
N	27	28	19	20

^aP > 0.05 for one-way ANOVA with post-hoc Tukey HSD test.

Patch-Clamp Electrophysiology.

Neuro-2a cells were visualized with an inverted microscope (IX71; Olympus, Tokyo, Japan) and green illumination (for visualizing fluorescent proteins). Whole-cell patch-clamp techniques were used with an Axopatch 200B amplifier (Molecular Devices Axon Instruments, Sunnyvale, CA), Digidata 1440A analog-to-digital converters (Molecular Devices Axon Instruments), and pClamp 10.3 software (Molecular Devices Axon Instruments). Data were sampled at 10 kHz and low-pass Bessel filtered at 2 kHz. Patch electrodes had a resistance of 2–6 MΩ when filled with intracellular solution. Series resistance (R_s) was compensated by 85%–95% throughout whole-cell patch-clamp recording, and data were discarded if the R_s exceeded 25 MΩ at the start or end of the recording.

Intracellular and extracellular solutions were used as in Henderson et al. (2016). The intracellular solution was as follows: 135 mM K-gluconate, 5 mM KCl, 5 mM EGTA, 0.5 mM CaCl₂, 10 mM HEPES, 2 mM Mg-ATP, and 0.1 mM GTP. The pH of the intracellular solution was adjusted to 7.2 with Tris-base, and osmolarity was adjusted to 298 mOsmol with sucrose. Just prior to gigaseal formation, the junction potential between the patch pipette and bath solutions was nulled. Chronic menthol and/or nicotine treatments were 24–30 hours long and began 24 hours after transfection. All recordings were performed 47–58 hours following the start of transfection (average of 51.75–52.75 hours across all treatments) (Tables 1–3).

Acetylcholine was dissolved in extracellular solution containing the following: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose (280–320 mOsmol, pH set to 7.3 with Tris-base). The 1 mM Zn²⁺ solutions were made from zinc acetate stock solutions, as in a previous study that tested Zn²⁺ sensitivity of rat α3β4 nAChRs (Hsiao et al., 2001).

For both [ACh]-response and Zn²⁺-inhibition experiments, ACh and/or 1 mM Zn²⁺ were applied by local laminar flow using an Octaflow II perfusion system [0.5–5 seconds; 6 psi; the Octaflow micromanifold tip (200 μm internal diameter) was located ∼1.0 mm from the Neuro-2a cell] (ALA Scientific Instruments, Farmingdale, NY) onto voltage-clamped Neuro-2a cells (holding potential of −65 mV, after correcting for a junction potential of −16 mV). Actual current growth and decay times exceeded our calculated solution exchange time of ∼18 milliseconds, probably because of deviations from laminar flow. Extracellular solution was perfused over the entire recording chamber at ∼2.7 chamber volumes/min, while extracellular solution was also continuously perfused by local laminar flow at 2 psi (for 30 seconds to 5 minutes) from the Octaflow II perfusion system when ACh and/or Zn²⁺ were not being perfused. In the experiments, the nicotine- or menthol-containing medium was replaced by several washes with extracellular solution free of both nicotine and menthol, over an average period of 1.7 and 1.8 hours, respectively (minimum of 30 minutes). In experiments on Neuro-2a and other cultured mammalian cells, within 2 seconds after nicotine is removed from the extracellular solution, the intracellular solution [nicotine] falls to undetectable levels (Shivange et al., 2019); this rules out retention of nicotine intracellularly (Jia et al., 2003). We estimate that extracellular [nicotine] or [menthol] solution decreased to <1 fM. Recordings were then commenced.

For [ACh]-response experiments, to avoid receptor desensitization from repetitive ACh application, we applied ACh at up to 5-minute intervals. There was no statistically significant current rundown in n ≥ 4 Neuro-2a cells transfected with mouse α3-GFP β4 nAChRs with three ACh applications at (duration of ACh application): 10 µM (5 seconds) at 2-minute intervals; 50 µM (5 seconds) at 3-minute intervals; 100 µM (2 seconds) at 3-minute intervals; 500 µM (1 second) at 5-minute intervals; and 1000 µM (0.5 seconds) at 5-minute intervals [P > 0.05, one-way ANOVA with post-hoc Tukey honestly significant difference (HSD) test]. Also, current rundown caused by 1 µM ACh applied for the 5-second duration was ruled out by observing no significant difference between responses of 100 µM ACh applied 3 minutes before and 30 seconds after this 1 µM ACh application (P > 0.05, two-tailed t test). Therefore, when collecting [ACh]-response data, ACh was applied at concentrations, durations, and time intervals that do not cause significant current rundown (i.e., 200 µM ACh was applied for 1 second and allowed to recover for 5 minutes before the next ACh application, because 500 µM ACh applied for 1 second at 5-minute intervals did not cause current rundown as explained previously). Up to six out of the nine ACh concentrations (1, 3, 10, 20, 50, 100, 200, 500, and 1000 µM ACh) were applied to the cell in each recording session, and 100 µM ACh was applied twice in each recording session as a measure of current rundown. Peak current amplitudes were normalized to 1 for the maximum response for each recording session. The ACh concentrations were applied in a different order when recording at different cells. Data from different recording sessions were combined to form mean [ACh]-response curves for different chronic treatments. [ACh]-response data were fitted in Origin 2018 software (OriginLab Corporation, Northampton, MA) by

(1)

where y = response; A1 = minimum response (usually near 0); A2 = maximum response (near 1); p = Hill coefficient; and x = [ACh].

To compare desensitization between control and 500 nM chronic (−)-menthol treatments, we calculated the percentage of current decay from the ACh-evoked peak at 1700 and 650 milliseconds from the start of the ACh-evoked growth phase for 100 and 200 µM ACh applications, respectively.

For Zn²⁺-inhibition experiments, 100 µM ACh (with or without 1 mM Zn²⁺) was applied for 1 second at 2.5-minute intervals, and 1 mM Zn²⁺ and 100 µM ACh were coapplied in between the 100 µM ACh only applications. Immediately prior to coapplication of 1 mM Zn²⁺ and 100 µM ACh, 1 mM Zn²⁺ (without ACh) was applied to the cell for 2 seconds by local laminar flow at 6 psi from the Octaflow II perfusion system. For each Zn²⁺-inhibition experiment, there were four 100 µM ACh only applications and three 1 mM Zn²⁺ and 100 µM ACh coapplications. The percentage of Zn²⁺ inhibition for each experiment was calculated by

(2)

where x = mean 100 µM ACh peak current amplitude in the presence of 1 mM Zn²⁺, and y = mean 100 µM ACh peak current amplitude in the absence of 1 mM Zn²⁺.

For both [ACh]-response and Zn²⁺-inhibition experiments, data were discarded for individual recordings showing substantial current rundown (a >2-fold difference in current amplitude evoked by 100 µM ACh applications). Neither menthol nor nicotine were present during any ACh applications in this study. Before the cell was recorded, the chronic nicotine and menthol treatments ended at average times of 1.7 and 1.8 hours, respectively.

Data Analyses.

Clampfit 10.3 (Molecular Devices Axon Instruments) was used to analyze peak current amplitudes. In our figures, we moved electrophysiological traces along the time axis to account for variations in flow rates from the Octaflow manifold. Because peak inward current amplitudes of transfected α3β4 nAChRs in our data set and published data often exceed –4 nA (Krashia et al., 2010), recorded peak current amplitudes were further corrected for underestimation due to uncompensated R_s (5%–15%) by assuming a linear current-voltage relationship and a reversal potential of 0 mV. Uncompensated R_s was multiplied by current amplitude to calculate the shift in membrane potential (V_m). The shift in V_m allowed us to estimate the size of underestimated current amplitudes [(shift in V_m/holding V_m of −65 mV) × current amplitude]. Bar graphs, [ACh]-response curve fitting, and average 100 μM ACh and 200 μM ACh waveforms were completed and plotted in Origin 2018 (OriginLab Corportion). We performed statistical tests (one-way ANOVA with post-hoc Tukey HSD test and two-tailed t tests) using Origin 2018 software (OriginLab Corportion) and/or Microsoft Excel (Redmond, WA). Mean ± S.E. values are represented in bar charts, average waveforms, and tables (Figs. 1–3; Tables 1–3).

Results

Chronic Menthol Treatment Does Not Alter the Stoichiometry of Functional Mouse α3β4 nAChRs.

To help understand how menthol in mentholated cigarettes may influence nicotine dependence, we studied how the functional characteristics of mouse α3β4 nAChRs are altered by chronic (24–30 hours) 500 nM menthol treatment. In Neuro-2a cells transiently transfected with mouse α3-GFP and β4 nAChR subunits at a 1:1 ratio, we studied whole-cell patch-clamp currents. This Neuro-2a cell system has proven amenable for studies of chronic nicotine and/or chronic menthol treatment effects on α4β2 and α6β2 nAChRs (Srinivasan et al., 2011, 2012b; Henderson et al., 2014, 2017; Henderson and Lester, 2015). Some clonal cell types express transfected membrane proteins at levels so high that aspects of subunit assembly, membrane tracking, and turnover become limiting, distorting regulatory processes that occur in neurons (Dávila-García et al., 1999; Xiao and Kellar, 2004; Lomazzo et al., 2011). Neuro-2a cells have more modest expression levels than various HEK293-derived cell lines, allowing good control of membrane proteins (Moss et al., 2009). In previous studies of Neuro-2a cells transfected with α4β2 and α6β2 nAChRs, varying the ratio of transfected subunits does affect the stoichiometry of the nAChRs (Srinivasan et al., 2012a; Fox et al., 2015)—and exposure to chronic nicotine does upregulate nAChRs (Srinivasan et al., 2011, 2012a,b; Fox et al., 2015) and exposure to menthol alone also upregulates nAChRs (Henderson et al., 2016).

From our experiments, the EC₅₀ for ACh is 99 μM (n = 6–13) (Fig. 1, A and B). Our EC₅₀ is similar to the EC₅₀ of 92 μM for ACh at Xenopus oocytes injected with mouse α3-GFP and β4 nAChR-encoding complementary RNAs (obtained from the same cDNA constructs used for our experiments) at a 2:3 ratio, respectively (Shih et al., 2014). Furthermore, our EC₅₀ value for ACh at mouse α3-GFP and β4 nAChRs transfected in Neuro-2a cells is consistent with EC₅₀ values for ACh at WT and modified mouse, rat, and human α3 and β4 nAChRs encoding complementary RNAs injected into oocytes, as well as for human WT α3 and β4 nAChRs encoding complementary DNAs transfected at a 1:1 ratio in HEK293 cells (Wang et al., 1998; Drenan et al., 2008; Grishin et al., 2010; Krashia et al., 2010).

Zn²⁺-Inhibition of ACh-Evoked Currents at Mouse α3-GFP β4 nAChRs Following Chronic Menthol and/or Nicotine Treatment.

Because our ACh-induced currents in transfected mouse α3β4 nAChRs and transfected human nAChRs α3β4s have similar EC₅₀ values and waveforms as previously published data (Krashia et al., 2010), we hypothesized that we would find similar effects of Zn²⁺ on 100 μM ACh at mouse and human nAChRs α3β4s. We employed Zn²⁺ to assess stoichiometry. Moreover, synaptic vesicles of forebrain neurons contain Zn²⁺ (Frederickson et al., 2000). Zn²⁺ is released from neurons calcium and depolarization dependently, reaching estimated transient concentrations of almost 300 μM (Assaf and Chung, 1984; Howell et al., 1984).

Krashia et al. (2010) assessed the effects of Zn²⁺ on ACh-evoked currents at human α3β4 nAChRs of different stoichiometries. Krashia et al. (2010) showed that a wide range of [Zn²⁺] differentially affected the two α3β4 nAChR stoichiometries. Within this range, 1 mM Zn²⁺ actually produced opposite effects on ACh-evoked currents at the two populations, enhancing (α3)₂(β4)₃ but inhibiting (α3)₃(β4)₂ (Krashia et al., 2010). Therefore, we chose 1 mM Zn²⁺ as a sensitive way to determine mouse α3β4 nAChR stoichiometry changes by chronic drug treatments. If chronic menthol treatment (with or without co-chronic nicotine treatment) shifts toward the (α3)₃(β4)₂ stoichiometry, we expect greater levels of inhibition by 1 mM Zn²⁺ compared with no chronic menthol treatment (with or without co-chronic nicotine treatment, respectively). Using western blotting with coimmunoprecipitation of total cell lysates, Mazzo et al. (2013) found that 1 mM nicotine treatment of 24 hours causes a shift (62%) toward (α3)₂(β4)₃ from the (α3)₃(β4)₂ stoichiometry in transfected HeLa cells. We used a lower concentration of 250 μM nicotine in our experiments to avoid potential nonspecific effects caused by alkaline pH shifts with 1 mM nicotine. Nicotine concentrations of similar range (hundreds of micromolars) exist transiently in the airway surface liquid of smokers (Clunes et al., 2008; Mazzo et al., 2013), and α3β4 nAChRs may enhance the growth of lung cancers (Improgo et al., 2010, 2013). Therefore, the nicotine concentration we study in our experiments has pharmacological relevance. Neither of the chronic treatments significantly changed the cell capacitance relative to control treatment (P > 0.05 for one-way ANOVA with post-hoc Tukey HSD test) (Table 1).

In the absence of nicotine, chronic 500 nM (−)-menthol treatment of 24–30 hours did not significantly alter the percentage of 1 mM Zn²⁺-inhibition of 100 μM ACh-evoked currents compared with control treatment (no menthol and no nicotine) (P > 0.05 for one-way ANOVA with post-hoc Tukey HSD test, 48.5% ± 4.8% vs. 50.1% ± 6.5%, n = 16 and 14, respectively) (Fig. 1, C and D; Table 1). In addition, neither chronic 250 μM nicotine alone (48.7% ± 6.1%, n = 19), nor combined chronic 250 μM nicotine and 500 nM (−)-menthol (55.4% ± 5.3%, n = 20) treatments significantly altered the level of 1 mM Zn²⁺-inhibition of 100 μM ACh-evoked current compared with control treatment (P > 0.05 for both treatments for one-way ANOVA with post-hoc Tukey HSD test) (Fig. 1, C and D; Table 1). This result indicates that compared with control treatment, neither of the chronic treatments [(−)-menthol alone, nicotine alone, or (−)-menthol and nicotine combined] changes the stoichiometry of mouse α3β4 nAChRs. Also, combined chronic 250 μM nicotine and 500 nM (−)-menthol treatment did not significantly alter the level of 1 mM Zn²⁺ inhibition of 100 μM ACh-evoked current compared with chronic 250 μM nicotine treatment alone (55.4% ± 5.3% vs. 48.7% ± 6.1%; P > 0.05 for one-way ANOVA with post-hoc Tukey HSD test; n = 20 and 19, respectively) (Fig. 1, C and D; Table 1). This result emphasizes that chronic 500 nM (−)-menthol does not influence 1 mM Zn²⁺ sensitivity of ACh-evoked currents at mouse α3β4 nAChRs. Because these control, chronically menthol-treated, and/or chronically nicotine-treated α3β4 nAChRs all showed >45% inhibition by 1 mM Zn²⁺ (Fig. 1, C and D; Table 1), we suggest that most mouse α3β4 nAChRs in these transfected Neuro-2a cells possess the (α3)₃(β4)₂ stoichiometry. If these α3β4 nAChRs under the different chronic treatments were predominantly in the (α3)₂(β4)₃ stoichiometry, ACh-evoked currents would show potentiation in the presence of 1 mM Zn²⁺ (Krashia et al., 2010). Our suggestion is also consistent with the Krashia et al. (2010) study, which strongly suggested that HEK293 cells transfected with α3 and β4 nAChR subunits at a 1:1 ratio predominantly possess the (α3)₃(β4)₂ stoichiometry. Krashia et al. (2010) suggested this predominant (α3)₃(β4)₂ stoichiometry after observing greater shifts in the EC₅₀ of ACh in patch-clamp recorded transfected cells containing mutant α3 and WT β4 compared with cells containing WT α3 and mutant β4 nAChR constructs.

[ACh]-Response at Mouse α3-GFP β4 nAChRs Following Chronic Menthol Treatment.

As another experiment to probe stoichiometry changes by chronic menthol, we assessed the [ACh]-response relationship at mouse α3-GFP β4 nAChRs following chronic menthol treatment. Previous studies have shown how populations of different stoichiometries vary in the ACh EC₅₀ values by 2- to 3-fold at human α3β4 nAChRs (Krashia et al., 2010) and 7- to 8-fold at rat α3β4 nAChRs (Grishin et al., 2010). Because Krashia et al. (2010) found that Zn²⁺ inhibition is sensitive to α3β4 nAChR stoichiometry, our previous Zn²⁺-inhibition experiments do present strong evidence that chronic exposure to (−)-menthol and/or nicotine fails to affect α3β4 nAChR stoichiometry. However, we recently reported that (−)-menthol and (+)-menthol have markedly different effects in chronic exposure at α4β2 nAChRs (http://www.eneuro.org/content/eneuro/early/2018/12/20/ENEURO.0465-18.2018.full.pdf). Furthermore, cigarette manufacturers could consider responding to possible governmental bans on cigarettes containing (−)-menthol (https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm625884.htm), which is now the predominant isoform in mentholated cigarettes, by adding (+)-menthol instead. Therefore, we extended the experiments to determine whether chronic exposures to other forms of menthol [(±)-menthol or (+)-menthol] alter α3β4 nAChR stoichiometry. Neither of the chronic menthol treatments significantly changed the cell capacitance relative to control treatment (P > 0.05 for one-way ANOVA with post-hoc Tukey HSD test) (Table 2).

TABLE 2.

[ACh] response at mouse α3β4 nAChRs under different chronic (24–30 h) treatments

Cells were studied 51.75–52.75 h after the start of transfection and 25.75–26.5 h after the start of incubation in no drug-containing media or after menthol was added (both durations are averages). Mean ± S.E. values are represented in the table.

Chronic Treatment	Control	(−)-Menthol	(±)-Menthol	(+)-Menthol
N	6–13	5–12	4–11	6–13
EC50 (µM)	99 ± 14	134 ± 18	117 ± 16	142 ± 5
Hill slope	1.14 ± 0.18	1.42 ± 0.24	1.28 ± 0.21	1.59 ± 0.07
Cell capacitance (pF)a	19.7 ± 1.9	20.0 ± 3.7	17.6 ± 2.0	16.0 ± 1.9
Current density (pA/pF) of 100 µM ACh currentsa	−153.2 ± 33.3	−117.1 ± 41.4	−196.9 ± 42.9	−214.5 ± 33.4
Current density (pA/pF) of 1 mM ACh currentsa	−363.1 ± 84.6	−295.9 ± 86.7	−440.5 ± 64.1	−566.1 ± 84.4

^aP > 0.05 for one-way ANOVA with post-hoc Tukey HSD test.

Chronic treatments with either 500 nM (−)-menthol (EC₅₀ = 134 μM; n = 5–12), 500 nM (±)-menthol (EC₅₀ = 117 μM; n = 4–11), or 500 nM (+)-menthol (EC₅₀ = 142 μM; n = 6–13) for 24–30 hours did not shift the EC₅₀ value of ACh by >1.5-fold compared with control treatment (no menthol and no nicotine; EC₅₀ = 99 μM; n = 6–13) at Neuro-2a cells transfected with mouse α3-GFP and WT β4 nAChR subunits (Fig. 2, A and B; Table 2). These [ACh]-response experiments together with the Zn²⁺-inhibition experiments suggest that chronic menthol treatment does not shift the stoichiometry of functional mouse α3β4 nAChRs. The [ACh] response of control and chronic menthol-treated cells have Hill slope values between 1 and 2 (Table 2), as similarly reported in the published literature for α3β4 nAChRs (Grishin et al., 2010; Krashia et al., 2010; Shih et al., 2014). Furthermore, compared with control treatment, the different chronic menthol treatments did not significantly change the current density (pA/pF) of 100 μM ACh-evoked currents or 1 mM ACh-evoked currents (P > 0.05 for one-way ANOVA with post-hoc Tukey HSD test in both cases) (Table 2).

Fig. 2.

Chronic (24–30 hours) menthol treatment does not shift the [ACh]-response relationship at mouse α3β4 nAChRs transfected into Neuro-2a cells, but accelerates desensitization kinetics. Menthol and/or nicotine were not present during ACh application. Chronic treatments key: control = black; (−)-menthol = green; (±)-menthol = blue; and (+)-menthol = purple. (A) [ACh]-response curves (average normalized response ± S.E. values are represented in the curves) were constructed for chronic treatments with 500 nM (−)-menthol, 500 nM (+)-menthol, and 500 nM (±)-menthol (n = 5–12, 6–13, and 4–11, respectively, for the different concentrations) and plotted with control (no menthol, n = 6–13, as in Fig. 1). EC₅₀ values are given in Table 2. (B) Exemplar traces from chronic 500 nM (−)-menthol treatment displayed by their ACh concentration and duration of application. (C and D). Chronic menthol treatment alters desensitization of 100 μM (C) and 200 μM (D) ACh-evoked currents. The percentage of current decay from the ACh-evoked peak was calculated at 1700 and 650 ms from the start of the ACh-evoked growth phase for the 100 and 200 μM ACh applications, respectively. Mean current waveform curves with S.E. for no drug treated, and (−)-menthol, (+)-menthol, and (±)-menthol treated chronically at transfected Neuro-2a cells [average of 12, 12, 11, and 13 cells for 100 μM ACh (C), and average of 6, 6, 5, and 6 cells for 200 μM ACh (D), respectively]. Mean ± S.E. values are represented in the waveforms in (C and D).

The operational definition of desensitization is a decline in agonist-induced conductance while the agonist is present. Ton et al. (2015) have previously shown that acute menthol application accelerates desensitization of currents at the α3β4 nAChR by accessing the open state of the channel, and our experiments with chronic menthol treatment in the absence of acutely applied menthol and prior to agonist activation may highlight another mechanism for modulating desensitization. We assessed effects of chronic menthol exposure on desensitization. Compared with control treatment, chronic treatment with 500 nM (−)-menthol significantly accelerated desensitization of 100 μM ACh-evoked currents at mouse α3-GFP β4 nAChRs [P < 0.05 for the two-tailed t test, 39.8% ± 3.5% (n = 12) vs. 52.7% ± 5.1% (n = 12) current decay for control and chronic (−)-menthol treatment, respectively, at 1700 milliseconds from the start of the ACh-evoked growth phase] (Fig. 2C). Also, compared with control treatment, chronic treatment with 500 nM (−)-menthol significantly accelerated desensitization of 200 μM ACh-evoked currents at mouse α3-GFP β4 nAChRs [P < 0.01 for the two-tailed t test, 21.6% ± 3.4% (n = 6) vs. 45.1% ± 5.7% (n = 6) current decay for control and chronic (−)-menthol treatment, respectively, at 650 milliseconds from the start of the ACh-evoked growth phase] (Fig. 2D). Our results follow the known pattern that, at higher agonist concentrations, nAChR currents desensitize more rapidly. We also tested whether, for a given ACh concentration, higher agonist-induced currents desensitize more rapidly. We pooled data for 100 μM ACh-evoked currents, and separately, for 200 μM ACh-evoked currents, for the four chronic treatments [control, (−)-menthol, (±)-menthol, and (+)-menthol], with a criterion of >100 pA/pF to eliminate noisy contribution from relatively small signals. We compared the peak current density (pA/pF) and percentage of desensitization at 1700 and 650 milliseconds after the start of the growth phase [for 100 μM (n = 29) and 200 μM ACh-evoked currents (n = 20), respectively]. For both the 100 and 200 μM ACh-evoked current data sets, we found no significant Pearson’s correlation coefficient between peak current density and desensitization (ANOVA).

Chronic Nicotine But Not Chronic Menthol Treatment Reduces Functional PM Mouse α3β4 nAChR Levels.

We analyzed our data set to assess functional PM levels of mouse α3β4 nAChRs following chronic treatments with 500 nM (−)-menthol, 250 μM nicotine alone, combined 250 μM nicotine and 500 nM (−)-menthol, and control (neither menthol nor nicotine). The current density (peak current amplitude in pA/cell capacitance in pF) is an appropriate metric for functional PM levels of the mouse α3β4 nAChR. Unlike quantitative reverse-transcription polymerase chain reaction or western blot, whole-cell patch-clamp electrophysiology can reveal alterations in functional nAChR PM levels or alterations from post-translational changes. Neither of the chronic treatments in this current density analysis significantly changed the cell capacitance relative to control treatment (P > 0.05 for one-way ANOVA with post-hoc Tukey HSD test) (Table 3). In this section, we report on the results of t tests as we consider the subtle differences discussed subsequently as important findings in the field of regulation of nAChRs.

Chronic (−)-menthol treatment alone has no statistically significant effect on the current density of mouse α3β4 nAChRs compared with control treatment (no chronic nicotine or menthol) (P > 0.05 for both one-way ANOVA with post-hoc Tukey HSD test and two-tailed t test; −102.7 ± 19.6 pA/pF vs. −129.7 ± 22.9 pA/pF, n = 28 and 27, respectively) (Fig. 3; Table 3). Furthermore, combined chronic (−)-menthol and nicotine treatment had no statistically significant effect on the current density of mouse α3β4 nAChRs compared with chronic nicotine treatment alone (P > 0.05 for both one way-ANOVA with post-hoc Tukey HSD test and two-tailed t test; −71.9 ± 12.3 pA/pF vs. −62.7 ± 10.4 pA/pF, n = 20 and 19, respectively) (Fig. 3; Table 3). Interestingly, compared with control treatment (no chronic nicotine or menthol), both chronic 250 μM nicotine treatment alone (P < 0.05 by two tailed t test, P > 0.05 by one-way ANOVA with post-hoc Tukey HSD test; −129.7 ± 22.9 pA/pF vs. −62.7 ± 10.4 pF/pA, n = 27 and 19, respectively; 52% decrease) and combined chronic 250 μM nicotine and 500 nM menthol treatment (P < 0.05 by two tailed t test, P > 0.05 by one way-ANOVA with post-hoc Tukey HSD test; −129.7 ± 22.9 pA/pF vs. −71.9 ± 12.3 pA/pF, n = 27 and 20, respectively; 45% decrease) caused a reduction in the current density of mouse α3β4 nAChRs (Fig. 3; Table 3). Therefore, chronic nicotine treatment at 250 μM causes a reduction in functional mouse α3β4 nAChR PM levels in Neuro-2a cells. Because we extensively washed the nicotine (250 µM) for ≥30 minutes, and usually 100 minutes, in nicotine-free extracellular solution before the recording session (see Materials and Methods), for that cell no classically defined desensitization process would adequately describe the reduced function.

Discussion

We undertook these experiments to understand whether chronic exposure to submicromolar menthol alters the properties of α3β4 nAChRs, whose abundance in the medial habenula-interpeduncular pathway may dominate the aversive properties of nicotine. The pharmacokinetics of menthol in humans have proven challenging to study, presumably because menthol is glucuronidated via first-pass metabolism (Gelal et al., 1999), but we have estimated elsewhere that menthol concentrations in the brain of a mouse model of smoking are 0.5–2.5 μM (Henderson et al., 2016, 2017).

Long-term pharmacological effects on PM levels of nAChRs proceed, at least in part, via differential trafficking of the major subunit stoichiometries, α₂β₃ versus α₃β₂. Therefore, we sought to measure both subunit stoichiometry and PM nAChR levels. Chronic (24–30 hours) menthol treatment at 500 nM neither significantly altered subunit stoichiometry [(α3)₂(β4)₃ vs. (α3)₃(β4)₂] on the PM nor significantly changed functionally measured PM protein levels of mouse nAChR α3β4 (P > 0.05 for both).

Faced with this insensitivity to chronic menthol, we sought to know whether chronic nicotine itself affects either functional PM stoichiometry or functional PM protein levels in mouse α3β4 nAChR levels in the Neuro-2a cell assay system. Surprisingly, chronic nicotine at a concentration many times that found in the blood following smoking decreased rather than increased the current density of 100 μM ACh-evoked currents, indicating reduced functional mouse α3β4 PM protein levels.

The β subunit is probably the dominant factor causing the contrast between chronic nicotine and/or menthol effects on α4β2 and α6β2, versus on α3β4. In our assay system, α4β4 nAChRs are exported efficiently from the ER to the PM; most α4β2 nAChRs are not (Richards et al., 2011; Srinivasan et al., 2011). That is, in the absence of nicotine, mouse α4β4 nAChRs are already highly distributed in the PM relative to the ER. Previous experiments have shown a mechanistic basis for this difference. The mouse nAChR β4 subunit has an ER export motif (LXM), but no ER retention motif (RRQR), and these properties explain how mouse α3β4 nAChRs efficiently exit the ER to reach the PM (Srinivasan et al., 2011; Mazzo et al., 2013). Indeed, the crucial interaction may occur at the single previously nonbinding β subunit, also termed accessory (Crespi et al., 2018b). Mouse α4-eGFP β2 nAChRs with modified β2 subunits containing an export motif and without the ER retention motif were strongly localized to the PM relative to the ER (2.36-fold increase in PM-integrated density over mouse α4-eGFP WT β2 nAChRs), and the chronic (48 hours) nicotine treatment at 100 nM caused only a modest (1.2-fold) additional increase in the PM integrated density (Srinivasan et al., 2011). However, the chronic (48 hours) nicotine treatment at 100 nM upregulated mouse α4-GFP WT β2 nAChRs more substantially (1.9-fold increase in PM-integrated density) (Srinivasan et al., 2011). The export motif is absent in both the nAChR α3 subunit and mouse nAChR β2 subunit, while the mouse nAChR β4 subunit contains an ER export motif (Srinivasan et al., 2011; Mazzo et al., 2013). Therefore, it is mechanistically understandable how mouse nAChR α3β4 nAChRs have a high PM versus ER distribution without additional aids to ER export of α3β4 nAChRs.

What is the relevance to chronic effects of menthol? Previous experiments show that chronic submicromolar menthol also apparently acts in the early exocytotic pathway to aid the ER exit of nAChRs (Henderson et al., 2016, 2017). The observed details of chronic menthol effects differ from those of chronic nicotine effects, and probably also from the acute blocking effects of menthol at [menthol] >100-fold higher than our chronically applied concentrations (Ton et al., 2015). We tentatively suggested that chronic submicromolar menthol could act as a nonspecific chemical chaperone for α4β2 and α6β2 nAChRs (Henderson et al., 2016); however, the target could also be another protein in the early exocytotic pathway. In any case, because α3β4 nAChRs do not experience a rate-limiting step in the early exocytotic pathway due to the absence of an ER retention motif, it is mechanistically understandable that menthol treatment causes no further increase in the already high existing PM versus ER distribution.

Certain more subtle effects of chronic submicromolar menthol cannot be ruled out. Chronic 500 nM (−)-menthol treatment accelerated desensitization of ACh-evoked currents at mouse α3β4 nAChRs, even >1 hour after we washed out the menthol. This effect is unlikely to arise from a menthol-nAChR binding with a lifetime of ∼1 hour. We can also rule out sequelae of (−)-menthol interactions with TRPM8, the classic menthol target, because Neuro-2a cells do not express TRPM8 mRNA (Henderson et al., 2016). However, phosphorylation of nAChRs does, in some cases, enhance desensitization (Huganir et al., 1986; Hopfield et al., 1988; Nishizaki and Sumikawa, 1998; Di Angelantonio et al., 2011). Whether an unknown pathway activates protein kinase(s) during chronic exposure to 500 nM (−)-menthol, and whether nAChR phosphorylation is stable for >1 hour after the menthol is removed, cannot be evaluated at the present time.

The steps leading to functional α3β4 nAChRs reaching the cell membrane include: 1) assembly of subunits into a pentamer; and 2) post-assembly trafficking, which can be affected by degradation of the pentamer. While our experiments and previously published data indicate that α3 and β4 subunits preferentially assemble into a (α3)₃(β4)₂ stoiochiometry (Krashia et al., 2010), this stoichiometry is still prone to degradation (Mazzo et al., 2013). Therefore, our patch-clamp experiments mainly record currents from the (α3)₃(β4)₂ stoichiometry because the (α3)₂(β4)₃ stoichiometry may reach the plasma membrane less efficiently due to possibly lower pentamer assembly. Additional intracellular (α3)₃(β4)₂ complexes formed in the presence of chronic menthol may undergo degradation before they can be trafficked to the PM. Preliminary data from our laboratory lend support for such a mechanism, but at higher menthol concentrations than used here (Patowary et al., 2016).

Mechanism of Downregulation by Chronic Nicotine.

Previous experiments indicate that chronic treatments with nicotine have less dramatic effects on protein levels of nAChR α3β4 compared with nAChR α4β2 in several brain regions studied (Wang et al., 1998; Meyer et al., 2001; Dávila-García et al., 2003; Nguyen et al., 2003; Fox et al., 2015; Marks et al., 2015). In a contrasting report, chronic (14 days) nicotine treatment in rats caused downregulation of nAChR α3β4-like binding sites in the subiculum and cerebellum (Nguyen et al., 2003). Although efficient membrane trafficking usually limits further increases in the PM levels of α3β4 nAChRs, Mazzo et al. (2013) skillfully rendered membrane tracking the rate-limiting step by inhibiting protein synthesis with cycloheximide. Under these circumstances, nicotine-induced upregulation of human α3β4 nAChRs occurs through the increased stability of the (α3)₂(β4)₃ stoichiometry (Mazzo et al., 2013), leading to increased trafficking to the PM. It is unlikely that chronic nicotine treatment would cause decreased current density of 100 μM ACh-evoked currents through a shift toward the lower potency mouse (α3)₃(β4)₂ nAChR stoichiometry because: 1) when Neuro-2a cells in our study or HEK293 cells in another study were transfected with α3 and β4 nAChRs at a 1:1 ratio, the level of Zn²⁺-inhibition of ACh-evoked currents suggests (α3)₃(β4)₂ as the predominant stoichiometry (Krashia et al., 2010); and 2) if nicotine shifts α3β4 nAChR stoichiometry, it would be toward (α3)₂(β4)₃ (Mazzo et al., 2013), since nicotine shifting toward a stoichiometry of three α and two β subunits of any nAChR has not been previously reported.

Our finding that nicotine-induced downregulation of mouse α3β4 nAChRs is not common, but recalls experiments on other nAChRs. In toxin binding studies in rodents or primates, nicotine downregulates α6* nAChRs in some cases (Lai et al., 2005; McCallum et al., 2006b; Mugnaini et al., 2006) but not others (McCallum et al., 2006a; Visanji et al., 2006), and the effect depends on the detailed stoichiometry of the complex (Perez et al., 2008; Fox et al., 2015).

Further Questions and Conclusions.

It will be interesting to study whether chronic menthol treatment has different effects at α3β4 versus α3β4α5 nAChRs, analogous to the differential modulation by lynx1 of human α3β4 and α3β4α5 nAChRs (George et al., 2017). Furthermore, functional α3β4β3 nAChRs are present in the brain (Grady et al., 2009). It remains possible that the subunit in the accessory position influences the nature of the subunit interfaces (Walsh et al., 2018), and hence potentially the effects from chronic treatments by menthol or other chaperones.

In conclusion, chronic menthol treatment (500 nM, 24–30 hours) failed to shift the [ACh]-response relationship and Zn²⁺ sensitivity of ACh-evoked currents at mouse α3β4 nAChRs, suggesting no change in receptor stoichiometry at the PM. Furthermore, compared with no drug treatment, the current density of 100 ACh-evoked currents was not significantly changed following chronic menthol treatment (P > 0.05), indicating that functional mouse α3β4 nAChR PM levels were not changed. Mechanistically, these data are broadly consistent with the view that chronic effects of submicromolar menthol act via events in the early exocytotic pathway. Pathopharmacologically, our data sets suggest that smoking mentholated cigarettes, which enhances smoking addiction and nicotine addiction, exerts these effects via mechanisms other than chronic submicromolar exposure of α3β4 nAChRs.

Abbreviations

ACh

acetylcholine

ER

endoplasmic reticulum

GFP

green fluorescent protein

HSD

honestly significant difference

nAChR

nicotinic acetylcholine receptor

Neuro-2a

neuroblastoma-2a

PM

plasma membrane

R_s

series resistance

V_m

membrane potential

WT

wild type

Authorship Contributions

Participated in research design: Bavan, Henderson, Lester.

Conducted experiments: Bavan, Kim.

Performed data analysis: Bavan, Henderson, Lester.

Wrote or contributed to the writing of the manuscript: Bavan, Kim, Henderson, Lester.