Menthol stereoisomers exhibit different effects on α4β2 nAChR upregulation and dopamine neuron spontaneous firing

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.Wethen 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.