TY - JOUR T1 - Menthol stereoisomers exhibit different effects on α4β2 nAChR upregulation and dopamine neuron spontaneous firing JF - eneuro JO - eNeuro DO - 10.1523/ENEURO.0465-18.2018 SP - ENEURO.0465-18.2018 AU - Brandon J. Henderson AU - Stephen Grant AU - Betty W. Chu AU - Rezvan Shahoei AU - Stephanie M. Huard AU - Shyam S. M. Saladi AU - Emad Tajkhorshid AU - Dennis A. Dougherty AU - Henry A. Lester Y1 - 2018/12/21 UR - http://www.eneuro.org/content/early/2018/12/20/ENEURO.0465-18.2018.abstract N2 - 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 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 wildtype 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, sub µM 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.Significance Statement Menthol is the most popular flavor used in all forms of tobacco products. Depending on the method used to produce the menthol, these products contain two stereoisomers ((−)-menthol and (+)-menthol) in unregulated and poorly characterized amounts. We studied how these isomers of menthol differ in their ability to alter nicotinic receptors on dopamine neurons that contribute to tobacco addiction. Long-term treatment with (−)-menthol and (+)-menthol resulted in a stereospecific effect on nicotinic receptor upregulation and dopamine neuron excitability: (+)-menthol exhibited no effect while (−)-menthol exhibited a robust effect. In acute treatments, we found only slight differences between effects of the two isomers on nicotine receptors. This suggests that long-term exposure to (−)-menthol may contribute to exacerbating tobacco addiction. ER -