Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the ß(+)/α(-) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3ß2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-ß2D190, involving the C loop region of the ß2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-ß2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-ß2A127 and α3E173-ß2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation.

Intra-subunit flexibility underlies activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

Allosteric modulation is a general feature of nicotinic acetylcholine receptors, yet the structural components and movements important for conversions among functional states are not well understood. In this study, we examine the communication between the binding sites for agonist and the modulator morantel (Mor) of neuronal α3ß2 receptors, measuring evoked currents of receptors expressed in Xenopus oocytes with the two-electrode voltage-clamp method. We hypothesized that movement along an interface of ß sheets connecting the agonist and modulator sites is necessary for allosteric modulation. To address this, we created pairs of substituted cysteines that span the cleft formed where the outer ß sheet meets the ß sheet constituting the (-)-face of the α3 subunit; the three pairs were L158C-A179C, L158C-G181C and L158C-K183C. Employing a disulfide trapping approach in which bonds are formed between neighboring cysteines under oxidation conditions, we found that oxidation treatments decreased the amplitude of currents evoked by either the agonist (ACh) or co-applied agonist and modulator (ACh + Mor), by as much as 51%, consistent with the introduced bond decreasing channel efficacy. Reduction treatment increased evoked currents up to 89%. The magnitude of the oxidation effects depended on whether agonists were present during oxidation and on the cysteine pair. Additionally, the cysteine mutations themselves decreased Mor potentiation, implicating these residues in modulation. Our findings suggest that these ß sheets in the α3 subunit move with respect to each other during activation and modulation, and the residues studied highlight the contribution of this intramolecular allosteric pathway to receptor function.

Specificity determinants of allosteric modulation in the neuronal nicotinic acetylcholine receptor: a fine line between inhibition and potentiation.

We are interested in the allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs). We have postulated that the anthelmintic morantel (Mor) positively modulates (potentiates) rat α3ß2 receptors through a site located at the ß(+)/α(-) interface that is homologous to the canonical agonist site (J Neurosci 29:8734-8742, 2009). On this basis, we aimed to determine the site specificity by studying differences in modulation between α3ß2 and α4ß2 receptors. We also compared modulation by Mor with that of the related compound oxantel (Oxa). Whereas Mor and Oxa each potentiated α3ß2 receptors 2-fold at saturating acetylcholine (ACh) concentrations, Mor had no effect on α4ß2 receptors, and Oxa inhibited ACh-evoked responses. The inhibition was noncompetitive, but not due to open channel block. Furthermore, the nature and extent of modulation did not depend on subunit stoichiometry. We studied six positions at the α(-) interface that differ between α3 and α4. Two positions (α3Ile57 and α3Thr115) help mediate the effects of the modulators but do not seem to contribute to specificity. Mutations in two others (α3Leu107 and α3Ile117) yielded receptors with appreciable α4-character; that is, Mor potentiation was reduced compared with wild-type α3ß2 control and Oxa inhibition was evident. A fifth position (α3Glu113) was unique in that it discriminated between the two compounds, showing no change in Mor potentiation from control but substantial Oxa inhibition. Our work has implications for rational drug design for nicotinic receptors and sheds light on mechanisms of allosteric modulation in nAChRs, especially the subtle differences between potentiation and inhibition.

The positive allosteric modulator morantel binds at noncanonical subunit interfaces of neuronal nicotinic acetylcholine receptors.

We are interested in the positive allosteric modulation of neuronal nicotinic acetylcholine (ACh) receptors and have recently shown that the anthelmintic compound morantel potentiates by enhancing channel gating of the alpha3beta2 subtype. Based on the demonstration that morantel-elicited currents were inhibited by the classic ACh competitor dihydro-beta-erythroidine in a noncompetitive manner and that morantel still potentiates at saturating concentrations of agonist (Wu et al., 2008), we hypothesized that morantel binds at the noncanonical beta2(+)/alpha3(-) subunit interface. In the present study, we created seven cysteine-substituted subunits by site-directed mutagenesis, choosing residues in the putative morantel binding site with the aid of structural homology models. We coexpressed the mutant subunits and their respective wild-type partners in Xenopus oocytes and characterized the morantel potentiation of ACh-evoked currents, as well as morantel-evoked currents, before and after treatment with a variety of methanethiosulfonate (MTS)-based compounds, using voltage-clamp recordings. The properties of four of the seven mutants, two residues on each side of the interface, were changed by MTS treatments. Coapplication with ACh enhanced the extent of MTS modification for alpha3A106Cbeta2 and alpha3beta2S192C receptors. The activities of two mutants, alpha3T115Cbeta2 and alpha3beta2T150C, were dramatically altered by MTS modification. For alpha3beta2T150C, while peak current amplitudes were reduced, potentiation was enhanced. For alpha3T115Cbeta2, both current amplitudes and potentiation were reduced. MTS modification and morantel were mutually inhibitory: MTS treatment decreased morantel-evoked currents and morantel decreased the rate of MTS modification. We conclude that the four residues showing MTS effects contribute to the morantel binding site.

Morantel allosterically enhances channel gating of neuronal nicotinic acetylcholine alpha 3 beta 2 receptors.

We studied allosteric potentiation of rat alpha3beta2 neuronal nicotinic acetylcholine receptors (nAChRs) by the anthelmintic compound morantel. Macroscopic currents evoked by acetylcholine (ACh) from nAChRs expressed in Xenopus laevis oocytes increase up to 8-fold in the presence of low concentrations of morantel (< or =10 microM); the magnitude of the potentiation depends on both agonist and modulator concentrations. It is noteworthy that the potentiated currents exceed the maximum currents achieved by saturating (millimolar) concentrations of agonist. Studies of macroscopic currents elicited by prolonged drug applications (100-300 s) indicate that morantel does not increase alpha3beta2 receptor activity by reducing slow (> or =1 s) desensitization. Instead, using outside-out patch-clamp recordings, we demonstrate that morantel increases the frequency of single-channel openings and alters the bursting characteristics of the openings in a manner consistent with enhanced channel gating; these results quantitatively explain the macroscopic current potentiation. Morantel is a very weak agonist alone, but we show that the classic competitive antagonist dihydro-beta-erythroidine inhibits morantel-evoked currents noncompetitively, indicating that morantel does not bind to the canonical ACh binding sites.

Single-channel properties of N- and L-subtypes of acetylcholine receptor in Ascaris suum.

We are interested in the properties of the target site of cholinergic anti-nematodal drugs for therapeutic reasons. The target receptors are ligand-gated ion channels that have different subtypes, and each subtype may have a different pharmacology. In a contraction assay using the parasitic nematode Ascaris suum, our laboratory has identified several subtypes, including an N-subtype, preferentially activated by nicotine, and an L-subtype, preferentially activated by levamisole. Here we use patch-clamp recordings to test the hypothesis that the single-channel selectivities of nicotine and levamisole are different. Unitary currents evoked by nicotine in this preparation were characterised for the first time. In some patches, both nicotine and levamisole activated small- and large-conductance channels. In other patches, the agonists activated just one channel amplitude. Discriminant analysis allowed classification of the one-conductance patch channels into the small or large categories, based on sets defined by the two-conductance patch data. The small channels had a conductance of 26.1+/-1.5 pS, n=18 (mean+/-SEM); the large conductance channels had a conductance of 38.8+/-1.2 pS, n=23 (mean+/-SEM). Analysis of amplitude histograms of the two-conductance patches showed that nicotine preferentially activated the small-conductance channels and levamisole preferentially activated the large-conductance channels. Our observations suggest that the N-subtype receptor channel has a conductance of 26 pS channel and the L-subtype receptor channel has a conductance of 39 pS.

The anthelmintic levamisole is an allosteric modulator of human neuronal nicotinic acetylcholine receptors.

L-[-]-2,3,5,6-Tetrahydro-6-phenylimidazo[2,1b]-thiazole hydrochloride (levamisole) is an anthelmintic that targets the nicotinic acetylcholine receptors of parasitic nematodes. We report here the effects of levamisole on human neuronal alpha 3 beta 2 and alpha 3 beta 4 nicotinic receptors, heterologously expressed in Xenopus oocytes and studied with the voltage clamp method. Applied alone, levamisole was a very weak partial agonist for the two subunit combinations. When co-applied with acetylcholine, micromolar concentrations of levamisole potentiated responses, while millimolar concentrations inhibited them; these effects were complex functions of both acetylcholine and levamisole concentrations. The differences in the levamisole effects on the two receptor combinations suggest that the effects are mediated by the beta subunit. Several combinations of agonist and anthelmintic gave the dual potentiation/inhibition behavior, suggesting that the modulatory effects are general. Levamisole inhibition showed macroscopic characteristics of open channel block. Several results led us to conclude that levamisole potentiation occurs through noncompetitive binding to the receptor. We propose pseudo-site binding for noncompetitive potentiation by levamisole.