Menthol Binding and Inhibition of a7-Nicotinic Acetylcholine Receptors

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner

Menthol Inhibits 5-HT 3

The effects of alcohol monoterpene menthol, a major active in-gredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 mM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited bymenthol in a concentration-dependent (IC505 163mM)manner. The effects of menthol developed gradually, reaching a steady-state level within 10–15 minutes and did not involve G-proteins, since GTPgS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (2), (1), and racemic menthol inhibited 5-HT3 receptor–mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid injections an

Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+)-dependent Cl(-) channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing Ba(2+). Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125)I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+) transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner

Menthol Inhibits 5-Ht3 Receptor-Mediated Currents

The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 mu M)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 mu M) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTP gamma S activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy) ethane-N,N,N\u27,N\u27-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [H-3]GR65630 was not altered in the presence of menthol (up to 1 mu M), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 mu M). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors

Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+)-dependent Cl(-) channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing Ba(2+). Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125)I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+) transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner

Menthol Inhibits 5-HT 3 Receptor-Mediated Currents

ABSTRACT The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT 3 ) receptors expressed in Xenopus laevis oocytes. 5-HT (1 mM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC 50 5 163 mM) manner. The effects of menthol developed gradually, reaching a steadystate level within 10-15 minutes and did not involve G-proteins, since GTPgS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (2), (1), and racemic menthol inhibited 5-HT 3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(oaminophenoxy)ethane-N,N,N9,N9-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT 3 antagonist [ 3 H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT 3 receptor. Finally, 5-HT 3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 mM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT 3 receptors

A multiple sequence alignment and conservation scores obtained with MUSCLE Vr.3.8.31 [30] between the human GABA_AR-α₁ subunit (UniProt AC: P14867), human α7-nACHR (UniProt AC:P36544 and the muscle nACh receptor subunit chain A ([24]; PDB ID: 2BG9) in (A).

<p>The fragment highlights, through the boxed and shaded region, key residue positions from the M3 segment of GABA_AR-α₁ evaluated by Williams and Akabas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067674#pone.0067674-Williams1" target="_blank">[29]</a> for propofol binding. The docking simulation indicates binding of the menthol ligand on muscle nACh receptor residue THR292. This position is indicated by the red triangle and corresponds to the most frequent docking site. High sequence conservation about the binding site with muscle nACh receptor could indicate similar binding site characteristics for α7-nACh receptor. (<b>B</b>) Representative docked configuration for menthol (ZINC ID: 01482164) on the crystal structure of muscle nACh receptor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067674#pone.0067674-Miyazawa1" target="_blank">[24]</a>; PDB ID: 2BG9). 1) Top-down view of nACh receptor with chain A colored in blue, B in red, C in gray, D in orange, and E in yellow. The binding site for the ligand is circled. As the α7-nACh receptor is a homopentamer, this conserved binding site could also be found on all five receptor subunits of the functional receptor. 2) Side-view of the circled binding site. 3) Lowest-energy (−6.15 kcal/mol) configuration, on four of the ten simulations resulting in lowest interaction energies, is shown for the L-menthol ligand. The h-bond that stabilizes the ligand onto the crystal structure is formed at residue THR292 of the muscle nACh receptor chain A (blue) at a distance of 2.21 <u>Å</u>. 4) The second most-frequent configuration for the ligand, corresponding to interaction energy of −5.98 kcal/mol, obtained on two of the top ten simulations. An h-bond with residue LEU250 of chain A (blue) at a distance of 1.97 <u>Å</u> stabilizes this docked configuration.</p

Inhibition of acetylcholine-induced currents by menthol is independent of the activation of pertussis toxin sensitive receptors, membrane potential and Ca²⁺-activated Cl⁻ channels.

<p>(<b>A</b>) Bar presentation of the effects of 30 µM menthol application (15 min) on the maximal amplitudes of ACh-induced currents in oocytes injected with 50 nl distilled-water, controls (n = 5) or 50 nl of PTX (50 µg/ml, n = 6). Bars represent the means ± S.E.M. (<b>B</b>) α₇-nicotinic acetylcholine receptor expressing oocytes injected with 50 nl distilled water and recorded in 2 mM Ca²⁺ containing MBS solution (control) or injected with 50 nl of BAPTA (100 mM) and recorded in 2 mM Ba²⁺ containing MBS solution (BAPTA). Bars represent the means ± S.E.M. of 6 to 8 oocytes. The numbers of oocytes are presented on top of each bar. There was no statistically significant difference between menthol (30 µM) inhibition in the presence and in the absence of BAPTA injections (<i>P</i>>0.05, n = 5–8, ANOVA). (<b>C</b>) Current-voltage relationships of acetylcholine-activated currents in the absence and presence of menthol (30 µM). Normalized currents activated by 100 µM acetylcholine before (control,•) and after 15 min treatment with menthol (○). Each data point presents the normalized means and S.E.M. of five to six oocytes. (<b>D</b>) Quantitative evaluation of the effect of menthol as percent inhibition at different voltages.</p