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

Abstract

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.

The Impact of Electronic Nicotine Delivery System (ENDS) Flavors on Nicotinic Acetylcholine Receptors and Nicotine Addiction-Related Behaviors

“…In separate investigations, menthol was also determined to act as a noncompetitive antagonist on both α7 and α3β4* nAChRs [ 167 , 168 ]. A combination of computational docking, site-directed mutagenesis, and electrophysiology revealed that menthol may exert its effect on nAChRs by binding to the 9′ position in the transmembrane 2 (M2) helix of nAChRs [ 22 ] ( Figure 4 A). Here, a series of mutations at the 9′ leucine residues (L9′) of the high-sensitivity α4β2 nAChRs were made and characterized with electrophysiology to investigate the connection between menthol’s pharmacology and the L9′ site [ 22 ].…”

“…A combination of computational docking, site-directed mutagenesis, and electrophysiology revealed that menthol may exert its effect on nAChRs by binding to the 9′ position in the transmembrane 2 (M2) helix of nAChRs [ 22 ] ( Figure 4 A). Here, a series of mutations at the 9′ leucine residues (L9′) of the high-sensitivity α4β2 nAChRs were made and characterized with electrophysiology to investigate the connection between menthol’s pharmacology and the L9′ site [ 22 ]. It was concluded that menthol’s inhibitory actions rely on the presence of the L9′ site of the M2 helix and only one menthol molecule is sufficient for α4β2 nAChR inhibition.…”

“…Here, it is important to distinguish the fact that the concentrations of menthol used to examine its inhibitory activity on nAChRs is several orders of magnitude higher (>30 µM) than what is considered smoking/vaping-relevant (<2 µM). The studies that have used low concentrations of menthol (0.5 µM) have determined that menthol combined with nicotine does enhance nAChR upregulation on VTA dopamine neurons and also enhances the excitability of these same neurons (discussed further below) [ 18 , 22 ]. A previously published review has extensively disseminated the recent investigations that have elucidated the mechanism of menthol’s actions on nicotine reward and reinforcement [ 158 ].…”

“…Despite this gap in knowledge, there are numerous reports of menthol’s effect on nicotine addiction, including menthol’s ability to enhance nicotine reward and reinforcement [ 16 , 17 , 18 , 19 ]. These effects are due to menthol-induced nicotinic acetylcholine receptor (nAChR) upregulation [ 18 , 20 , 21 ], enhanced dopamine neuron excitability and dopamine release [ 22 , 23 ], and TrpM8-dependent mechanisms [ 24 ]. Based on a recent study reporting green apple and other fruity flavors to be the most popular of ENDS flavorants [ 9 , 25 ], additional reports have identified popular green apple flavorants, farnesol and farnesene, to not only enhance nicotine reward in a mouse model, but also display rewarding properties in the absence of nicotine [ 19 , 26 , 27 ].…”

Flavor-specific enhancement of electronic cigarette liquid consumption and preference in mice

“…In addition, eliquids are available in many different flavors, and some of the most popular flavors among youth are mint, mango and fruit (Leventhal et al, 2019). The impact of different flavors is only beginning to be determined in preclinical models, with the majority of studies focusing on menthol (Alsharari et al, 2015;Wickham, 2015;Henderson et al, 2019).…”