Ashoor A et al. (2013), Menthol binding and inhibition of α7-nicotinic ...

XB-ART-47994

PLoS One 2013 Jan 01;87:e67674. doi: 10.1371/journal.pone.0067674.

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Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

Ashoor A , Nordman JC , Veltri D , Yang KH , Al Kury L , Shuba Y , Mahgoub M , Howarth FC , Sadek B , Shehu A , Kabbani N , Oz M .

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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.

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Species referenced: Xenopus laevis
Genes referenced: chrna4

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	Figure 2. Time and concentration-dependence of menthol inhibition of Î±7-nicotinic acetylcholine receptor-mediated ion currents.(A) Inhibition of the Î±7-nicotinic acetylcholine receptor increases with the prolongation of menthol pre-application time. Each data point represents the mean Â± S.E.M. of 5 to 6 oocytes. (B) Menthol inhibits Î±7-nicotinic acetylcholine receptor function in a concentration-dependent manner. Each data point represents the mean Â± S.E.M. of 7 to 9 oocytes. The curve is the best fit of the data to the logistic equation described in the methods section.
	Figure 3. Inhibition of acetylcholine-induced currents by menthol is independent of the activation of pertussis toxin sensitive receptors, membrane potential and Ca2+-activated Clâ channels.(A) Bar presentation of the effects of 30 ÂµM menthol application (15 min) on the maximal amplitudes of ACh-induced currents in oocytes injected with 50 nl distilled-water, controls (nâ=â5) or 50 nl of PTX (50 Âµg/ml, nâ=â6). Bars represent the means Â± S.E.M. (B) Î±7-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 Â± 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 ÂµM) inhibition in the presence and in the absence of BAPTA injections (P>0.05, nâ=â5â8, ANOVA). (C) Current-voltage relationships of acetylcholine-activated currents in the absence and presence of menthol (30 ÂµM). Normalized currents activated by 100 ÂµM acetylcholine before (control,â¢) and after 15 min treatment with menthol (â). Each data point presents the normalized means and S.E.M. of five to six oocytes. (D) Quantitative evaluation of the effect of menthol as percent inhibition at different voltages.
	Figure 4. Concentration-response curves for acetylcholine-induced currents and binding of [125I] Î±-bungarotoxin in control and in the presence of menthol.(A) Effect of menthol on the acetylcholine concentration-response relationship. Oocytes were voltage-clamped at â70 mV and currents were activated by applying acetylcholine (1 ÂµM to 3 mM). Oocytes were exposed to 100 ÂµM menthol for 15 min and acetylcholine was reapplied. Paired concentration-response curves were constructed and responses normalized to maximal response under control conditions. EC50 and slope values were determined by fitting the curves from 6 to 8 oocytes to the standard logistic equation as described in the methods section. Data points obtained before (control) and after 15 min treatment with menthol (100 ÂµM) were indicated by filled circles, open circles, and open triangles, respectively. Each data point presents the normalized means and S.E.M. of five to six experiments. (B) The effects of menthol on the specific binding of [125I] Î±-bungarotoxin to oocyte membrane preparations. In the presence and absence of menthol, specific binding as a function of the concentration of [125I] Î±-bungarotoxin is presented. Data points for controls and menthol (100 ÂµM) are indicated by filled circles, and open circles, respectively. Data points are the means of three independent experiments carried out in triplicate.
	Figure 5. Menthol attenuates nicotine-induced calcium signaling in neural cells.(A) A representative PC12 cell showing the expression of Î±7-nACh receptors in the soma and primary neurite. Green fluorescence: fBgtx labeling; red fluorescence: anti-rhodamine phalloidin immunostaining was used to determine ROI for calcium imaging. (B) Live cell imaging of cells expressing the genetically encoded calcium sensor GCaMP5G. ROI within soma and neurite shown via the orange and red dotted lines, respectively. Rows top to bottom: cells pre-treated with Control (0.3% ethanol); Menthol (30Î¼M); Î±-bungarotoxin (Bgtx) (50nM). Cellular images captured at 6/8/10/12 seconds (s). Nicotine (50Î¼M) was applied at 8-10s. (C) Average fluorescence signal data for soma and neurite ROI imaged for 20 seconds. nâ€Š=â€Š40 cells, **P>0.01â€.
	Figure 6. Menthol attenuates Î±7 nACh receptor calcium signaling.(A) Live cell imaging of cells expressing GCaMP5G. ROI within soma and neurite shown via the organge and red dotted line, respectively. ROI selection is based on co-detection of fBgtx and GCaMP5G as indicated in Fig. 5. Rows top to bottom: cells pre-treated with Control (0.3% ethanol); Menthol (30 ÂµM); Î±-bungarotoxin (Bgtx) (50 nM). Image frames captured at 6/8/10/12 seconds (s). PNU (10 ÂµM) was applied at 8â€“10 s. (B) Average fluorescence signal data for soma and neurite ROI imaged for 20 seconds. nâ€Š=â€Š40 cells, **P>0.01â€.
	Figure 7. A multiple sequence alignment and conservation scores obtained with MUSCLE Vr.3.8.31 [30] between the human GABAAR-Î±1 subunit (UniProt AC: P14867), human Î±7-nACHR (UniProt AC:P36544 and the muscle nACh receptor subunit chain A ([24]; PDB ID: 2BG9) in (A).The fragment highlights, through the boxed and shaded region, key residue positions from the M3 segment of GABAAR-Î±1 evaluated by Williams and Akabas [29] 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 Î±7-nACh receptor. (B) Representative docked configuration for menthol (ZINC ID: 01482164) on the crystal structure of muscle nACh receptor ([24]; 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 Î±7-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 (â6.15 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 Ã. 4) The second most-frequent configuration for the ligand, corresponding to interaction energy of â5.98 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 Ã stabilizes this docked configuration.
	Figure 1. Effect of menthol on Î±7-nicotinic acetylcholine receptor-mediated ion currents.(A) Records of currents activated by acetylcholine (ACh, 100 ÂµM) in control conditions (left), during co-application of 30 ÂµM menthol and acetylcholine after 10 min pretreatment with 30 ÂµM menthol (middle), and 15 min following menthol washout (right). (B) Time-course of the effect of menthol (100 ÂµM) on the peaks of the acetylcholine-induced currents. Each data point represents the normalized mean Â± S.E.M. of 4 to 5 oocytes. Duration of drug application is indicated by the horizontal bar in the figure. (C) Comparison of the extent of inhibition caused by 100 ÂµM of (+), (â), and racemic forms of menthol application for 15 min. Bars represent the means Â± S.E.M. from 6 to 7 cells. (D) Comparison of the effect of 30 ÂµM of racemic menthol application for 15 min on the currents activated by 100 ÂµM acetylcholine or 10 ÂµM nicotine. Bars represent the means Â± S.E.M. from 5 to 6 cells.

References [+] :

Ahijevych, Menthol pharmacology and its potential impact on cigarette smoking behavior. 2004, Pubmed