Henderson BJ et al. (2018), Menthol Stereoisomers Exhibit Different Effects...

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eNeuro 2018 Jan 01;56:. doi: 10.1523/ENEURO.0465-18.2018.

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Menthol Stereoisomers Exhibit Different Effects on α4β2 nAChR Upregulation and Dopamine Neuron Spontaneous Firing.

Henderson BJ , Grant S , Chu BW , Shahoei R , Huard SM , Saladi SSM , Tajkhorshid E , Dougherty DA , Lester HA .

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Menthol contributes to poor cessation rates among smokers, in part because menthol enhances nicotine reward and reinforcement. Mentholated tobacco products contain (-)-menthol and (+)-menthol, in varying proportions. We examined these two menthol stereoisomers for their ability to upregulate α4β2 nAChRs and to alter dopamine neuron firing frequency using long-term, low-dose (≤500 nm) exposure that is pharmacologically relevant to smoking. We found that (-)-menthol upregulates α4β2 nAChRs while (+)-menthol does not. We also found that (-)-menthol decreases dopamine neuron baseline firing and dopamine neuron excitability, while (+)-menthol exhibits no effect. We then examined both stereoisomers for their ability to inhibit α4β2 nAChR function at higher concentrations (>10 µm) using the Xenopus oocyte expression system. To probe for the potential binding site of menthol, we conducted flooding simulations and site-directed mutagenesis. We found that menthol likely binds to the 9´ position on the TM2 (transmembrane M2) helix. We found that menthol inhibition is dependent on the end-to-end distance of the side chain at the 9´ residue. Additionally, we have found that (-)-menthol is only modestly (∼25%) more potent than (+)-menthol at inhibiting wild-type α4β2 nAChRs and a series of L9´ mutant nAChRs. These data reveal that menthol exhibits a stereoselective effect on nAChRs and that the stereochemical effect is much greater for long-term, submicromolar exposure in mice than for acute, higher-level exposure. We hypothesize that of the two menthol stereoisomers, only (-)-menthol plays a role in enhancing nicotine reward through nAChRs on dopamine neurons.

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Genes referenced: ecd

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	Figure 1. Chronic exposure of menthol stereoisomers causes different effects on α4β2 nAChR upregulation. A, Representative TIRFM images of neuro-2a cells transfected with α4-SEP and β2 nAChR subunits. Menthol stereoisomers (500 nm) were added 24 h before imaging sessions. Scale bars, 10 µm. Each panel image shows representative cells at pH 7.4. B, PMRID was quantified for SEP nAChRs following treatment with menthol stereoisomers. For each condition, n > 30 cells. Data are the mean ± SEM. p < 0.05; *p < 0.01 (one-way ANOVA with Tukey). Two-way ANOVA, (+)-menthol vs (−)-menthol, F(1,381) = 30.34 and p < 0.0001. Exact p values are provided in the text.
	Figure 2. Menthol stereoisomers cause different effects on dopamine neuron baseline firing frequency. A1, B1, Representative image of a TH-positive cultured midbrain dopamine neuron (A1) and a TH-negative putative GABA neuron (B1). Scale bars, 20 µm. A2, B2, Representative waveforms of a TH-positive dopamine neuron with Ih (A2) and a TH-negative putative GABA neuron without Ih (B2). A3, B3, B4, Representative action potential from cultured dopamine and GABA neurons. C, Baseline firing frequency of TH-positive dopamine neurons treated with control, (+)-menthol, or (−)-menthol (500 nm, each) for 10 d. Data are the mean ± SEM. p < 0.05; *p < 0.01 (one way ANOVA with Tukey). Circles overlaid with bars represent individual recordings that constitute the mean value for each respective group (n = 17, 44, and 19 for control, (+)-menthol, and (−)-menthol, respectively). Exact p values are provided in the text. Full data are plotted as a scatterplot with mean ± SEM values plotted as an overlaid bar chart. D, Representative whole-cell current-clamp traces for TH-positive dopamine neurons treated with control or menthol stereoisomers. E1–E3, 9% of the (+)-menthol-treated dopamine neurons displayed dramatic variances in firing frequency. E2 and E3 are magnifications of blue and orange boxes, respectively, in E1.
	Figure 3. Chronic treatment with menthol stereoisomers causes different effects on dopamine neuron excitability. A1, A2, Representative image of TH-positive dopamine neuron with a diagram of the typical placement of patch and puffer pipets. B1–B3, Representative whole-cell current-clamp recordings from TH-positive dopamine neurons treated with control, (+)-menthol, or (−)-menthol (500 nm each) for 10 d. Arrows indicate a 300 ms application of 300 µm ACh to stimulate nAChRs. C1–C3, Mean firing frequency over time plot of TH-positive dopamine neurons before and after the ACh puff (indicated by arrow). C4, Quantification of firing frequency of dopamine neurons for the 3 s after ACh puff. Data are the mean ± SEM. n = 5–9 TH-positive dopamine neurons.
	Figure 4. Acute applications display only slight pharmacological differences among menthol stereoisomers. A, Concentration–response curves of ACh on oocytes injected with α4 and β2 nAChR subunits. Injections were biased to assemble high-sensitivity (α4)2(β2)3 nAChRs (1:10) or low-sensitivity (α4)3(β2)2) nAChRs (10:1). B1, B2, Concentration–response curve of menthol stereoisomers with high sensitivity (α4)2(β2)3 nAChRs (B1) or low sensitivity (α4)3(β2)2 nAChRs (B2). C1, C2, Concentration–response curve of ACh in the absence or presence of (−)-menthol or (+)-menthol (50 µm) with high sensitivity (α4)2(β2)3 nAChRs (C1) or low sensitivity (α4)3(β2)2 nAChRs (C2). Refer to Tables 1–3 for values for Hill coefficient, EC50, and IC50.
	Figure 5. Snapshots of a menthol molecule during flooding simulation. A, Menthol (shown in green sticks) is introduced in the extracellular solution. Menthol first encounters the extracellular domain (ECD) of the human α4β2 nicotinic receptor. B, Menthol binds to different residues on the ECD. C, Menthol reaches the “top” of the M2 region. D–F, For the rest of the flooding simulation, menthol remains in the transmembrane domain, between the “top” of the M2 region and the ring of 9´ Leu residues (shown as red sticks). Menthol does not move toward the cytoplasmic end of M2. The full movie may be downloaded from https://drive.google.com/drive/folders/1R7PWDpd7QpffZt7r9b3BWlH73WZgNbH0?usp=sharing.
	Figure 6. A snapshot of a menthol molecule sitting in the center of the ring of 9´ leucine residues. The carbons of menthol are shown in green, its hydrogens in white, and its oxygen in red. The carbons of the 9´ leucine residues are shown in gray, their oxygens in red, and their nitrogens in blue. The hydrogens of the leucine residue are not shown in the figure. The image is a higher-resolution snapshot from the movie available at https://drive.google.com/file/d/1eETqhvRk50-UayXNfUG94sHSwxSK6OoC/view.
	Figure 7. α4L9´ mutations probe the putative binding site of menthol. A1, A2, Concentration–response curves for menthol against (α4[L9´X])2(β2)3, where X is any amino acid. Each receptor is activated by its respective EC50 dose of ACh (A1) (−)-menthol and (A2) (+)-menthol. B, Concentration–response curves for (α4[L9´X])3(β2)2 and (α4)3(β2)2 using 1 µm ACh. C, Comparing the percentage maximum current induced when the oocyte is exposed to 100 µm menthol. p < 0.05; p < 0.01; p < 0.005; **p < 0.001. Exact p values are provided in extended data (Fig. 7-1). D1, Comparing the IC50 values. In this plot, (α4[L9´A])2(β2)3 is omitted because at no tested concentration of menthol was the receptor inhibited 50%. D2, Plot of IC30 values comparing (+)-menthol and (−)-menthol; n = 6–18 oocytes.
	Figure 8. A, The ratio of currents elicited by an EC50 concentration of ACh and 100 µm menthol over the current elicited by ACh alone. Error bars represent the SEM; n = 7-33 oocytes. B, Average Hill coefficients for wild-type and all mutant α4β2 mutants tested (p = 0.07). Error bars represent the SD; n = 7-39 mutants
	Figure 9. Potency of menthol depends on L9´ residue size. A1–B2, Plot of IC40 for (−)-menthol versus residue length (A1) or residue volume (B1) and plot of IC30 for (+)-menthol versus residue length (A2) or residue volume (B2). C1, Fitting the IC40 values for (−)-menthol against the reduced AA index. C2, Fitting the IC30 values for (+)-menthol against the reduced AA index. R 2 values in C1 and C2 for each parameter are indicated in Figure 9-1 and Figure 9-2. D, Concentration–response relationship for (−)-menthol on (α4[L9´I])3(β2)2 and (α4[L9´O-Me-Thr])3(β2)2 nAChRs. Structures of the two amino acids are shown to the left along with their end-to-end length; n = 7–18 oocytes.

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