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

Heteromeric α3β4 nAChRs are pentameric ligand-gated cation channels that include at least two α3 and at least two β4 subunits. They have functions in peripheral tissue, peripheral and central nervous systems. We examined the effects of chronic treatment with menthol, a major flavor additive in tobacco cigarettes and in electronic nicotine delivery systems, on mouse α3β4 nAChRs transiently transfected into neuroblastoma 2a (Neuro-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 Zn^(2+) 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 μM 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.