Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor

Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).     

Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.     

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.     

Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor

Interactions between lipids and the nicotinic acetylcholine receptor from Torpedo californica have been measured in reconstituted membranes containing purified receptor and defined lipids. The ability of brominated lipids to partially quench the intrinsic fluorescence of the acetylcholine receptor has been exploited to monitor contacts between the protein and the surrounding lipid. Relative binding constants for lipid binding to the protein have been quantitatively determined by measuring quenching observed in mixtures of brominated and nonbrominated lipids by use of equilibrium exchange equations developed by London and Feigenson [London, E., & Feigenson, G. W. (1981) Biochemistry 20, 1939-1948] and by Simmonds et al. [Simmonds, A. C., Rooney, E. K., & Lee, A. G. (1984) Biochemistry 23, 1432-1441]. Dioleoylphosphatidylcholine and its dibromo derivative are the two principal lipids used in the reconstituted membranes to establish the quenching parameters. Competition studies between cholesterol and phosphatidylcholine indicate that cholesterol does not compete effectively for the phospholipid sites presumed to surround the membrane-embedded portions of the receptor (annular lipids). However, dibromocholesterol partially quenches the receptor and leads to additional quenching of receptor in pure dibromophosphatidylcholine membranes. The results are consistent with the presence of additional binding sites for cholesterol that are not accessible to phospholipids (nonannular sites). Similar results are obtained by using cholesterol hemisuccinate and its dibromo analogue, both of which can be introduced into membranes more easily than cholesterol because of their greater solubility in water. Fatty acids appear to compete for both annular and nonannular sites, and analysis of the quenching data suggests that there are 5-10 nonannular sites associated with the receptor. Cholesterol has been shown to play a critical role in both acetylcholine receptor structural stabilization and ion channel activity, and the results presented here provide additional information about cholesterol-receptor interactions.     

Cembranoid and long-chain alkanol sites on the nicotinic acetylcholine receptor and their allosteric interaction

Long-chain alkanols are general anesthetics which can also act as uncharged noncompetitive inhibitors of the peripheral nicotinic acetylcholine receptor (AChR) by binding to one or more specific sites on the AChR. Cembranoids are naturally occurring, uncharged noncompetitive inhibitors of peripheral and neuronal AChRs, which have no demonstrable general anesthetic activity in vivo. In this study, [3H]tenocyclidine ([3H]TCP), an analogue of the cationic noncompetitive inhibitor phencyclidine (PCP), was used to characterize the cembranoid and long-chain alkanol sites on the desensitized Torpedo californica AChR and to investigate if these sites interact. These studies confirm that there is a single cembranoid site which sterically overlaps the [3H]TCP channel site. This cembranoid site probably also overlaps the sites for the cationic noncompetitive inhibitors, procaine and quinacrine. Evidence is also presented for one or more allosteric cembranoid sites which negatively modulate cembranoid affinity for the inhibitory site. In contrast, long-chain alkanols inhibit [3H]TCP binding through an allosteric mechanism involving two or more alkanol sites which display positive cooperativity toward each other. Double inhibitor studies show that the cembranoid inhibitory site and the alkanol sites are not independent of each other but interfere allosterically with each other's inhibition of [3H]TCP binding. The simplest models consistent with the observed data are presented and discussed.     

Identification of multiple allosteric sites on the M1 muscarinic acetylcholine receptor

Staurosporine and four staurosporine derivatives were docked on the rhodopsin-based homology model of the M1 muscarinic acetylcholine receptor in order to localize the possible allosteric sites of this receptor. It was found that there were three major allosteric sites, two of which are located at the extracellular face of the receptor, and one in the intracellular domain of the receptor. In the present study, the localization of these binding sites is described for the first time. The present study confirms the existence of multiple allosteric sites on the M1 muscarinic receptor, and lays the ground for further experimental and computational analysis to better understand how muscarinic receptors are modulated via their allosteric sites. These findings will also help to design and develop novel drugs acting as allosteric modulators of the M1 receptor, which can be used in the treatment of the Alzheimer's disease.     

Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic

Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[(3)H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 microm, 3-[(3)H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the alpha-subunit in the desensitized state. The incorporation into the alpha-subunit was mapped to two large proteolytic fragments. One fragment of approximately 20 kDa (alpha V8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of approximately 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within alpha V8-20, the primary site of incorporation was alpha Glu-262 at the C-terminal end of alpha M2, labeled preferentially in the desensitized state. The incorporation at alpha Glu-262 approached saturation between 1 microm, with approximately 6% labeled, and 275 microm, with approximately 30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at approximately 1% of the levels in alpha Glu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.     

Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state

The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two α subunits are rotated relative to its non-α subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the α subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the αG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the α subunits.     

Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel

Previous studies have established the presence of overlapping binding sites for the noncompetitive antagonists (NCAs) amobarbital, tetracaine, and 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine ([(125)I]TID) within the ion channel of the Torpedo nicotinic acetylcholine receptor (AChR) in the resting state. These well-characterized NCAs and competitive radioligand binding and photolabeling experiments were employed to better characterize the interaction of the dissociative anesthetics ketamine and thienylcycloexylpiperidine (TCP) with the resting AChR. Our experiments yielded what appear to be conflicting results: (i) both ketamine and TCP potentiated [(125)I]TID photoincorporation into AChR subunits; and (ii) ketamine and TCP had very little effect on [(14)C]amobarbital binding. Nevertheless, (iii) both ketamine and TCP completely displaced [(3)H]tetracaine binding (K(i)s approximately 20.9 and 2.0 microM, respectively) by a mutually exclusive mechanism. To reconcile these results we propose that, in the resting ion channel, TCP and ketamine bind to a site that is spatially distinct from the TID and barbiturate locus, while tetracaine bridges both binding sites.     

Multiple binding sites for phencyclidine on the nicotinic acetylcholine receptor from Torpedo ocellata electric organ

Binding of [3H]-phencyclidine ( [3H]-PCP) to acetylcholine-receptor enriched membrane from Torpedo ocellata electric organ was studied over a ligand concentration range of 1 to 200 microM. The results indicate that [3H]-PCP is bound to two classes of sites: high affinity (Kd = 6-9 microM) and low affinity (Kd = 85 microM) binding sites. In the absence of cholinergic drugs the ratio of high affinity [3H]-PCP binding sites to 125I-alpha-bungarotoxin (alpha-Bgt) binding sites is 0.37, and that of low affinity [3H]-PCP binding sites to 125I-alpha-Bgt is 1.06. Low affinity [3H]-PCP binding can be completely inhibited by alpha-bungarotoxin (alpha-Bgt), carbamylcholine and d-tubocurarine. This inhibition, together with the one to one stoichiometry with 125I-alpha-Bgt, suggests that the sites to which [3H]-PCP binds with low affinity are the acetylcholine (AcCho) binding sites. In the presence of 1 microM alpha-Bgt which blocks binding of [3H]-PCP to the AcCho binding sites, the ratio of high affinity [3H]-PCP sites to 125I-alpha-Bgt sites is 0.5, indicating the existence of one high affinity PCP site per receptor molecule, The toxin, however, decreases the apparent affinity of [3H]-PCP towards the AcCho receptor as well as the potency of tetracaine or dibucaine in inhibiting [3H]-PCP binding to that receptor. In the latter case the effect involves changes from a biphasic to a simple inhibition curve. The results suggest that non-competitive blockers to the AcCho receptors may affect their own sites as well, and that they do this also by binding to the AcCho binding sites. This is also inferred from the accelerated dissociation of [3H]-PCP from its high affinity binding sites by unlabeled PCP in the concentration range of 10(-3) to 10(-4) M, at which the drug occupies AcCho binding sites as well.     

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.     

Roles of agonist-binding sites in nicotinic acetylcholine receptor function

Under equilibrium conditions, the nicotinic acetylcholine receptor from Torpedo electroplax carries two high affinity-binding sites for agonists. It is generally assumed that these are the only agonist sites on the receptor and that their occupancy results in rapid channel activation followed by slower conformational transitions that lead to the high affinity equilibrium state. These slow transitions are thought to reflect the physiological process of desensitization. Here we show that preequilibration of the high affinity sites with saturating concentrations of carbamylcholine does not diminish the ion flux response to subsequent exposure to higher (activating) concentrations of this agonist. This finding has profound implications with respect to receptor function: (1) occupancy of the high affinity sites per se does not desensitize the receptor and (2) these sites cannot be directly involved in receptor activation. It is thus necessary to invoke the presence of additional binding sites in channel opening.     

Comparison of the toxin binding sites of the nicotinic acetylcholine receptor from Drosophila to human

Recombinant toxin binding proteins have been previously found to provide a convenient experimental system for the study of receptor-ligand recognition (Aronheim et al., 1988). Here, this system has been used to produce the binding sites of the cholinergic receptor derived from seven organisms, Torpedo californica, Xenopus, chick, mouse, calf, human, and Drosophila. These have been compared with respect to their toxin binding capacity. Scatchard analyses show that the KD values of alpha-bungarotoxin binding to the above sites are 63, 536, 150, 3200, 6200, 6470, and 1700 nM, respectively. These results reiterate the importance of alpha 183-204 as a ligand binding site. In order to increase the repertoire of sites available for study, chimeric structures were constructed. Through the analysis of such chimeras, some themes of the gross anatomy of the binding site can be learned. A positive subsite followed by a hydrophobic patch preceding a nucleophilic domain appears to be required for efficient toxin binding.     

Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain

The relationship between neuronal alpha-bungarotoxin binding proteins (alpha BGTBPs) and nicotinic acetylcholine receptor function in the brain of higher vertebrates has remained controversial for over a decade. Recently, the cDNAs for two homologous putative ligand binding subunits, designated alpha BGTBP alpha 1 and alpha BGTBP alpha 2, have been isolated on the basis of their homology to the N terminus of an alpha BGTBP purified from chick brain. In the present study, a panel of overlapping synthetic peptides corresponding to the complete chick brain alpha BGTBP alpha 1 subunit and residues 166-215 of the alpha BGTBP alpha 2 subunits were tested for their ability to bind 125I-alpha BGT. The sequence segments corresponding to alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were found to consistently and specifically bind 125I-alpha BGT. The ability of these peptides to bind alpha BGT was significantly decreased by reduction and alkylation of the Cys residues at positions 190/191, whereas oxidation had little effect on alpha BGT binding activity. The relative affinities for alpha BGT of the peptide sequences alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were compared with those of peptides corresponding to the sequence segments Torpedo alpha 1-(181-200) and chick muscle alpha 1-(179-198). In competition assays, the IC50 for alpha BGTBP alpha 1-(181-200) was 20-fold higher than that obtained for the other peptides (approximately 2 versus 40 microM). These results indicate that alpha BGTBP alpha 1 and alpha BGTBP alpha 2 are ligand binding subunits able to bind alpha BGT at sites homologous with nAChR alpha subunits and that these subunits may confer differential ligand binding properties on the two alpha BGTBP subtypes of which they are components.     

Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative

p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.     

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Fast kinetic studies on the allosteric interactions between acetylcholine receptor and local anesthetic binding sites.

Preincubation of receptor-rich membrane fragments from Torpedo marmorata with tertiary amine local anesthetics and several toxins such as histrionicotoxin, crotoxin and cerulotoxin, modifies the amplitude and time course of the relaxation processes monitored upon rapid mixing of the membrane fragments with the fluorescent agonist, Dns-C6-Cho. In particular, the amplitude of the rapid relaxation process, which is proportional to the fraction of acetylcholine receptor sites in a high-affinity state, increases; accordingly, the rate constant of the 'slow' and 'intermediate' relaxation processes also increases up to ten times (except with histrionicotoxin) whereas in a higher range of local anesthetic concentrations the rate constant of the 'rapid' relaxation process decreases. The data are accounted for by a two-state model of the acetylcholine regulator, assuming distinct binding sites for cholinergic agonists and local anesthetics and allosteric interactions between these two classes of sites; local anesthetics stabilize the regulator in a high-affinity state for agonists even in the absence of agonist, and modify the rate constants for th interconversions between the low-affinity and high-affinity states. The model accounts for the 'slow' fluorescence increase monitored upon addition of local anesthetics to a suspension of receptor-rich membranes supplemented with trace amounts of Dns-C6-Cho. The effect of local anesthetics on the apparent rate constant of the 'rapid' relaxation process can be accounted for on the basis of an additional low-affinity binding of local anesthetics to the acetylcholine receptor site. Finally the increase of the apparent rate constant of the 'intermediate' relaxation process can be simply accounted for by assuming the existence of a third state, corresponding to the 'active' state, to which local anesthetics bind and block ionic transport.     

Menthol suppresses nicotinic acetylcholine receptor functioning in sensory neurons via allosteric modulation

In this study, we have investigated how the function of native and recombinant nicotinic acetylcholine receptors (nAChRs) is modulated by the monoterpenoid alcohol from peppermint (-) menthol. In trigeminal neurons (TG), we found that nicotine (75 μM)-activated whole-cell currents through nAChRs were reversibly reduced by menthol in a concentration-dependent manner with an IC?? of 111 μM. To analyze the mechanism underlying menthol's action in more detail, we used single channel and whole-cell recordings from recombinant human α4β2 nAChR expressed in HEK tsA201 cells. Here, we found a shortening of channel open time and a prolongation of channel closed time, and an increase in single channel amplitude leading in summary to a reduction in single channel current. Furthermore, menthol did not affect nicotine's EC?? value for currents through recombinant human α4β2 nAChRs but caused a significant reduction in nicotine's efficacy. Taken together, these findings indicate that menthol is a negative allosteric modulator of nAChRs.