Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors

Abstract

The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

Fig. 1.

Effects of menthol on human α3β4 nAChR-mediated [Ca²⁺] influx and nicotine-stimulated ⁸⁶Rb⁺ efflux. (A) Representative Fluo-4 fluorescence images of α3β4-expressing HEK293 cells captured during control, repeated application of 30 μM ACh (upper images), or application of 30 μM ACh alone and ACh plus 100 μM menthol (lower images). (B, upper trace) Mean Fluo-4 fluorescence during repeated application of ACh (n = 39 cells) or (B, lower trace) ACh and ACh plus menthol (n = 37 cells). The interstimulus interval was 5 minutes. (C) Inhibition of nicotine-stimulated ⁸⁶Rb⁺ efflux in the presence of menthol or (D) 10-minute preincubation with menthol; n = 3 independent assays.

Fig. 2.

Menthol does not compete for the nAChR agonist binding site labeled by [³H]epibatidine. Membranes from human embryonic kidney cells expressing α3β4 nAChRs were incubated for 2 hours with ∼0.5 nM [³H]EB in the absence or presence of increasing concentrations of menthol or nicotine. The membranes were then filtered and counted. Data were analyzed by nonlinear least-squares regression analysis. The K_i of nicotine in these studies was 256 nM. Menthol at concentrations up to 1 mM did not compete for these receptors. Data shown are the mean ± S.E.M. of five independent assays.

Fig. 3.

Menthol enhances the decay of ACh-evoked currents. Representative inward currents in HEK293 cells expressing α3β4 nAChRs in response to (A) 30 μM ACh and (B) 100 μM, with or without menthol (100 μM) and after 60-second washout (A). The holding potential was –50 mV. (C) Menthol (100 μM) alone evokes no current. (D) A 5-minute treatment with menthol (100 μM) alone does not affect the subsequent current evoked by ACh (30 μM). (E) Summary of the peak current evoked by ACh and amount of current decay following 5-minute pretreatment with either control or menthol (n = 3). D, desensitization.

Fig. 4.

Menthol modulation of α3β4 nAChRs is dependent on the concentration of ACh. (A) Representative whole-cell inward currents in response to different concentrations of ACh in the absence (left) and in the presence (right) of 300 μM menthol in α3β4 nAChR-expressing cells. The current decay during desensitization was best fit to a one- or two-exponential function, yielding the indicated time constants. (B) Mean weighted average time constants for decay and (C) percent of desensitization (D) of the ACh-mediated current obtained in the absence of menthol (open circle) and in the presence of 300 μM of menthol (filled circle). Data are mean ± S.E.M., n = 3–5.

Fig. 5.

Menthol inhibits α3β4 nAChRs in a voltage- and use-independent manner and without affecting single-channel conductance. (A) Current voltage relationship for peak response to ACh (30 μM) and after 20-second application of menthol (100 μM). The background current in the absence of ACh is subtracted. The inset shows the fraction of the menthol versus control current at indicated potentials. (B) Responses to repeated, 5-second stimulation with ACh (30 μM) under control conditions (upper trace) or in the presence of 200 μM menthol (lower trace). Scale bars, 1 nA and 20 seconds. (C) Mean peak responses to ACh from experiments depicted in (B) (n = 3). (D) Representative single-channel currents from a cell-attached patch (V_M, –110 mV, 10 μM ACh in pipette) under control conditions (upper trace) and in the presence of 200 μM menthol. (E) All-points histograms constructed from 2 seconds of continuous data. The smooth lines represent best-fits to Gaussian functions yielding similar conductances (G) of 30.9 and 30.3 pS respectively. (F) Mean open time measured from data in (E) (measured from >50 events).

Fig. 6.

Concentration-dependent effects of menthol. (A) Representative currents activated by ACh (30 μM) and ACh plus menthol (30 and 300 μM); (B) Bar graph of time constant; (C) the fraction of desensitized (D) receptors; and (D) the current integrals in response to the concentration of menthol from 10 to 300 μM, which was coapplied with 30 μM ACh. Data are the normalized means ± S.E.M. of three to six experiments.

Fig. 7.

Menthol enhances the desensitization of the currents evoked by nicotine. Representative inward currents induced by (A) 1 μM, (B) 3 μM, (C) 10 μM, and (D) 100 μM nicotine in the absence of menthol (left), presence of 100 μM menthol (middle), and 5 minutes after washout of the menthol (right). Time constant (τ) and the extent of desensitization (D) were used to characterize the desensitization.

Fig. 8.

Menthol hinders the recovery of α3β4 nAChRs from desensitization. (A) Representative inward currents showing desensitization induced by coapplied 30 μM ACh and 100 μM menthol and the subsequent response to ACh following a 5-minute wash in either control bath solution (upper traces) or 100 μM menthol (lower traces). (B) Time course for recovery following the treatment described in (A), control (open circles), and menthol (closed circles). Data are the means ± S.E.M. of three to five experiments. (C) Menthol (100 μM) prevents recovery of α3β4 nAChRs following desensitization with 300 μM Ach, but receptors almost fully recover after 1 minute washout with control solution (n = 3).

Fig. 9.

Menthol inhibits ACh-evoked currents in nodose ganglia neurons. Representative current traces in a voltage-clamped neuron evoked by ACh (30 μM) and ACh plus menthol (100 μM). Note that menthol increases the speed and extent of desensitization. The cell was washed for 60 seconds between ACh applications.