Effects of Chronic Menthol at Alpha3Beta4 (α3β4)-Containing Nicotinic Acetylcholine Receptors

Abstract

Some heteropentameric nicotinic acetylcholine receptors (nAChRs) are up-regulated by chronic nicotine. Menthol is present in ∼ 30% of tobacco cigarettes sold in the United States. Compared with non-mentholated cigarettes, menthol-containing cigarettes are associated with reduced smoking cessation. Chronic menthol favors the lower sensitivity (α4)3(β2)2, whereas chronic nicotine favors the higher sensitivity (α4)2(β2)3 stoichiometry. Following chronic nicotine treatment, total cell lysates displayed a shift in stoichiometry towards (α3)2(β4)3 from (α3)3(β4)2. α3β4 nAChRs are highly expressed in the medial habenula and interpeduncular nuclei, which are involved in reward processing, and possibly nicotine addiction and withdrawal. We studied effects following chronic treatment (at least 24 hours) of 500 nM menthol at α3β4 nAChR using Forster resonance energy transfer (FRET), total internal reflection fluorescence microscopy (TIRFM), and whole-cell patch-clamp electrophysiology of Neuro-2a cells transiently expressing fluorescently labeled subunits. FRET experiments indicated a shift in stoichiometry toward (α3)3(β4)2 from (α3)2(β4)3. TIRFM experiments revealed α3 subunit up-regulation in the endoplasmic reticulum and α3β4 nAChR reduction in the plasma membrane. Our FRET experiments, however, include contributions from intracellular α3β4 nAChR stoichiometry. In contrast, patch clamp experiments measuring Zn2+ inhibition of acetylcholine-evoked currents indicate exclusively the functional cell surface α3β4 nAChR stoichiometry. Neither chronic menthol, chronic nicotine, nor combined chronic menthol and nicotine detectably alters Zn2+ inhibition of acetylcholine, showing that neither alters functional plasma membrane α3β4 nAChR stoichiometry. Furthermore, chronic menthol treatment shifts by < 1.5-fold the EC50 of acetylcholine at α3β4 nAChRs. These findings are consistent with our fluorescence-based experiments showing a reduction in endoplasmic reticulum exit sites following chronic menthol, which consequently reduces α3β4 nAChR delivery to the plasma membrane. Therefore, despite their intracellular effects, neither menthol nor nicotine influences cell surface α3β4 nAChR stoichiometry. Support: DA037743, DA036061, DA40047.