Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of α and non-α subunits, or homo-pentameric, composed of α7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between α1 and ? or α7 subunits. The replacement of M3 in α1 by ?M3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse ? chimeric subunit. The duration of the open state decreases with the increase in the number of α1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each α1M3 segment decreases the energy barrier of the closing process by ∼ 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of α1 sequence by α7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.     

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca²⁺ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [³H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6′) and valine (position 13′) rings, and (c) inhibits [³H]TCP, [³H]ibogaine, and [³H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.     

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.     

Insect nicotinic acetylcholine receptor: conserved neonicotinoid specificity of [(3)H]imidacloprid binding site

The insect nicotinic acetylcholine receptor (nAChR) is a major target for insecticide action. The rapidly expanding use of neonicotinoid insecticides of varied structures makes it increasingly important to define similarities and differences in their action, particularly for the first-generation chloropyridinyl compounds versus the second-generation chlorothiazolyl derivatives. We have shown with Musca domestica that a convenient and relevant determination of the neonicotinoid insecticide target is a binding site assay with [(3)H]imidacloprid ([(3)H]IMI). This study uses membranes from the aphids MYZUS: persicae and Aphis craccivora and from heads of the flies DROSOPHILA: melanogaster and Musca domestica to characterize the [(3)H]IMI binding sites relative to their number and possible species variation in structure-activity relationships. With emphasis on commercial neonicotinoids, six potent chloropyridinyl compounds are compared with the corresponding six chlorothiazolyl analogues (syntheses are given for chemicals prepared differently than previously described). The preference for chloropyridinyl versus chlorothiazolyl is not dependent on the insect species examined but instead on other structural features of the molecule. The chlorothiazolyl substituent generally confers higher potency in the clothianidin and desmethylthiamethoxam series and the chloropyridinyl moiety in the imidacloprid, thiacloprid, acetamiprid, and nitenpyram series. Two chlorothiazolyl compounds compete directly with the chloropyridinyl [(3)H]IMI for the same binding sites in MYZUS: and DROSOPHILA: membranes. This study shows conserved neonicotinoid specificity of the [(3)H]IMI binding site in each of the four insect species examined.     

Site-directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

The aim of this study was to present a new concept of site-directed reduction of disulfide bonds based upon the use of an affinity ligand harbouring a readily oxidizable dithiol. The cysteine bond involved in the acetylcholine binding site of the AChoR was specifically reduced by a carbamylcholine analogue. The ligand, in its oxidized form, was characterized by an affinity constant of 20 μM for the agonist binding site. In its dithiol form, it specifically reduced the disulfide between Cys-192 and Cys-193 on the -subunits of the nicotinic acetylcholine receptor. This reduction needed 10 times lower concentration when carried out with site-directed reducing agent (ARA) than with DTT, and was highly specific for the -subunits. The contribution of the carbamylcholine moiety of the site-directed reducing agent was clearly demonstrated in kinetic studies where reduction abilities of ARA, DTT and the methylated analogue of ARA (MeRA) were compared. At the same concentration (20 μM), DTT and MeRA had a 25 times lower initial rate of reduction than ARA. With 200 μM of DTT this initial reduction was still 4 times lower. Furthermore, the use of a maleimido undecagold cluster which specifically labeled the reduced nicotinic receptor opens the way to structural analysis of the agonist binding site by electron microscopy. These results demonstrate the potency of this kind of site-directed reducing agent for structural study of receptors or enzymes involving a disulfide bond in their active site.     

Antidepressants noncompetitively inhibit nicotinic acetylcholine receptor function

Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.     

Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor

We introduce the term ‘silent agonists’ to describe ligands that can place the α7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (5′-phenylanabaseine) was synthesized and identified as a new silent agonist for the α7 nAChR; it binds to the receptor but does not activate α7 nAChR channel opening when applied alone, and its agonism is revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus oocyte system. The concise synthesis was accomplished in three steps with the C–C bonds formed via Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative structural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrangement are needed to constitute one class of silent agonist for the α7 nAChR. Because silent agonists may act on signaling pathways not involving ion channel opening, this class of α7 nAChR ligands may constitute a new alternative for the development of α7 nAChR therapeutics.     

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).     

Multidimensional on-line screening for ligands to the alpha3beta4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase

The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.     

Interactions between tachykinins and diverse,human nicotinic acetylcholine receptor subtypes

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP) on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotinestimulated function measured using⁸⁶Rb⁺ efflux assays of human ganglionic (α3β4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC₅₀∼2.3 μM) or of human muscle-type (α1β1γδ) nAChR expressed in TE671/RD clonal cells (IC₅₀∼21 μM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC₅₀∼2.1 μM). Neurokinin A and eledoisin inhibit function (extrapolated IC₅₀ values between 60 and 160 μM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR is as sensitive as PC12 cell α3β4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of nAChR function, SP fails to affect binding of [³H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation of nervous system activity.     

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state NMR approach

Solid-state magic-angle spinning nuclear magnetic resonance (NMR) has sufficient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptor-toxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13C-,15N-labelled neurotoxin II. Despite the large size of the ligand-receptor complex ( > 290 kDa) and the high lipid content of the sample, we were able to detect and identify residues from the ligand. The comparison with solution NMR data of the free toxin indicates that its overall structure is very similar when bound to the receptor, but significant changes were observed for one isoleucine.     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

棉蚜乙酰胆碱受体亚基的选择性剪接和多重转录起始位点分析

乙酰胆碱受体在神经突触传导过程中具有重要作用,也是氯化胆碱类杀虫剂的作用靶标。采用RACE技术,成功地从棉蚜中克隆了3个nAChR亚基,其中2个为α亚型, 1个为β亚型,分别命名为Agα1、Agα2和Agβ1。通过锚定mRNA的5′mG结构, 5′RACE结果表明Agβ1有三个不同的剪接变体,具有不同长度的5′UTR区,表明Agβ1亚基具有多重的转录起始位点。其中,最短的剪接变体Agβ1C在蛋白编码区域也存在选择性剪接,位于D环区域的186 bp碱基缺失。3′RACE实验结果表明,Agα1亚基虽然具有ploy ( A)和加尾信号AATAAA等完整的mRNA基因结构,但缺失了终止子和乙酰胆碱受体α亚基保守的第4个跨膜区,文中对此做了进一步分析。分子进化树的分析表明,昆虫乙酰胆碱受体亚基应当被划分为三个不同的亚类群αⅠ,αⅡandβ。本文的研究揭示了昆虫乙酰胆碱受体亚基复杂的基因结构[动物学报51 (5) : 867 -878 , 2005]。     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [³H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 μM. These classes of sites exist in approximately equal numbers and all [³H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.     

Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor

Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace ³H-imidacloprid with a K_i value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC₅₀ of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism.Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.     

Assembly and trafficking of nicotinic acetylcholine receptors (Review)

Nicotinic acetylcholine receptors (nAChRs) are members of an extensive super-family of neurotransmitter-gated ion channels. In humans, nAChRs are expressed within the nervous system and at the neuromuscular junction and are important targets for pharmaceutical drug discovery. They are also the site of action for neuroactive pesticides in insects and other invertebrates. Nicotinic receptors are complex pentameric transmembrane proteins which are assembled from a large family of subunits; seventeen nAChR subunits (α1-α10, β1-β4, γ, δ and ε) have been identified in vertebrate species. This review will discuss nAChR subunit diversity and factors influencing receptor assembly and trafficking.     

Structural and functional studies of the nicotinic acetylcholine receptor by solid-state NMR

Over the last seven years, solid-state NMR has been widely employed to study structural and functional aspects of the nicotinic acetylcholine receptor. These studies have provided detailed structural information relating to both the ligand binding site and the transmembrane domain of the receptor. Studies of the ligand binding domain have elucidated the nature and the orientation of the pharmacophores responsible for the binding of the agonist acetylcholine within the agonist binding site. Analyses of small transmembrane fragments derived from the nicotinic acetylcholine receptor have also revealed the secondary structure and the orientation of these transmembrane domains. These experiments have expanded our understanding of the channels structural properties and are providing an insight into how they might be modulated by the surrounding lipid environment. In this article we review the advances in solid-state NMR applied to the nicotinic acetylcholine receptor and compare the results with recent electron diffraction and X-ray crystallographic studies.Presented at the Biophysical Society Meeting on Ion channels – from structure to disease held in May 2003, Rennes, France