Human Acetylcholine Receptor Research Articles

Human nicotinic acetylcholine receptor is a potential pharmacological target of oseltamivir

Oseltamivir (Tamiflu) effectively inhibits influenza virus-specific neuraminidase and therefore, is widely prescribed as an anti-influenza medication. Although a wide safety margin of oseltamivir has been reported, the possible neuronal adverse effects of this drug via unknown mechanisms are shown in some studies: dyskinesia, depressive episodes, hypothermia, and other CNS dysfunctions. We therefore, examined effects of oseltamivir on human nicotinic acetylcholine (ACh) receptors (nAChRs) with electrophysiological methods and found that oseltamivir reversely blocks nicotine- and ACh-evoked membrane currents in a concentration dependent manner in neuroblastoma cells derived from human peripheral neurons (IMR32) and in HEK cells expressing recombinant human α3β4 nAChRs. In contrast, an active metabolite of oseltamivir, oseltamivir carboxylate (OC) had little effect on the nicotine-evoked currents. Moreover, single channel analysis revealed that oseltamivir reduces the channel open time of nAChR without affecting the channel conductance. Our results demonstrate that human α3β4 nAChRs are a potential pharmacological target of oseltamivir, hence explaining a part of the adverse effects after ingestion of oseltamivir.

Direct proof of the in vivo pathogenic role of the AChR autoantibodies from myasthenia gravis patients.

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.

Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor, Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis

We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.

Expression of human AChR extracellular domain mutants with improved characteristics

The muscle nicotinic acetylcholine receptor (AChR) has a central role in neuromuscular transmission, and is the major target in the autoimmune disease myasthenia gravis (MG). We created mutants of the extracellular domains (ECDs) of the human α1, β1, δ and ɛ AChR subunits, whereby their Cys-loop was exchanged for that of the acetylcholine binding protein. The mutants were expressed in Pichia pastoris and had improved solubility resulting in 2- to 43-fold higher expression yields compared to the wild type. An additional mutant was created for the α1 ECD restoring its glycosylation site within the Cys-loop and its α-bungarotoxin binding ability. Furthermore, we constructed dimeric and pentameric concatamers of the mutant ECDs. All concatamers were successfully expressed as soluble secreted proteins, although the pentamers had about 10-fold lower expression than the dimers and were more susceptible to fragmentation. Initial crystallizations with the mutant ECDs were promising, and we reproducibly obtained crystals of the β1 ECD, diffracting at ∼12Å. Further optimization is underway to obtain crystals suitable for high resolution crystallography. The proteins described herein are useful tools in structural studies of the human muscle AChR and can be used in applications requiring high yields such as therapeutic adsorbents for MG autoantibodies.

Acute Activation, Desensitization and Smoldering Activation of Human Acetylcholine Receptors

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained “smoldering activation” occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)2β2 and (α4β2)2α4. The (α4β2)2β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)2α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

Proteomic investigation of human α7-nicotinic acetylcholine receptor signaling mechanisms

Proteomic investigation of human α7-nicotinic acetylcholine receptor signaling mechanisms

Functional chimera of the human α7 acetylcholine receptor for mechanistic investigations of allosteric modulation

Functional chimera of the human α7 acetylcholine receptor for mechanistic investigations of allosteric modulation

A ‘conovenomic’ analysis of the milked venom from the mollusk-hunting cone snail Conus textile—The pharmacological importance of post-translational modifications

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai’i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD50=34.2nMolkg−1. The non-post-translationally modified form, [Pro]2,8[Glu]16α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4±0.5μM. Interestingly its comparative PD50 (3.6μMolkg−1) in invertebrates was ∼100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.

Improved methodology to obtain large quantities of correctly folded recombinant N-terminal extracellular domain of the human muscle acetylcholine receptor for inducing experimental autoimmune myasthenia gravis in rats.

IntroductionHuman myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular system. Experimental autoimmune myasthenia gravis (EAMG) is a well-established animal model for MG that can be induced by active immunization with the Torpedo californica-derived acetylcholine receptor (AChR). Due to the expensive cost of purifying AChR from Torpedo californica, the development of an easier and more economical way of inducing EAMG remains critically needed.Material and methodsFull-length cDNA of the human skeletal muscle AChR α1 subunit was obtained from TE671 cells. The DNA fragment encoding the extracellular domain (ECD) was then amplified by polymerase chain reaction (PCR) and inserted into pET-16b. The reconstructed plasmid was transformed into the host strain BL21(DE3)pLysS, which was derived from Escherichia coli. Isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce the expression of the N-terminal ECD. The produced protein was purified with immobilized Ni2+ affinity chromatography and refolded by dialysis.ResultsThe recombinant protein was efficiently refolded to soluble active protein, which was verified by ELISA. After immunization with the recombinant ECD, all rats acquired clinical signs of EAMG. The titer of AChR antibodies in the serum was significantly higher in the EAMG group than in the control group, indicating successful induction of EAMG.ConclusionsWe describe an improved procedure for refolding recombinant ECD of human muscle AChR. This improvement allows for the generation of large quantities of correctly folded recombinant ECD of human muscle AChR, which provides for an easier and more economical way of inducing the animal model of MG.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Ų (1 Å=0.1 nm) of surface area, within which Arg³⁶ and Phe³² from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr¹⁸⁴ from loop-C of α7, while Asp³⁰ of α-btx forms a hydrogen bond with the hydroxy group of Tyr¹⁸⁴. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr¹⁸⁴ with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.

Fluoxetine prevents acetylcholine-induced excitotoxicity blocking human endplate acetylcholine receptor

Fluoxetine is an open channel blocker of fetal muscle acetylcholine (ACh) receptor (AChR) and slow-channel mutant AChRs. It is used commonly to treat patients with slow-channel congenital myasthenic syndromes. Fluoxetine effects on adult wild-type endplate AChR are less characterized, although muscle AChR isoforms are differentially modulated by some drugs. Excitotoxicity assays and patch clamp recordings were performed in human embryonic kidney 293 (HEK) cells expressing wild-type or slow-channel mutant human AChRs. Fluoxetine (2-10 μM) abolished ACh-induced death and decreased ACh-activated whole-cell currents in cells expressing all AChR types. In outside-out patches, fluoxetine rapidly curtailed ACh evoked unitary activity and macroscopic currents. The effect was increased if fluoxetine was applied before ACh. Fluoxetine is an open channel blocker, but it also affects AChR in the closed state. AChR blockade likely underlies the rescue of HEK cells from ACh-induced death.

English

Abstract Introduction The interaction of (S,S)and (R,R)N,6-dimethyltricyclo[5.2.1.0 2,6] decan-2-amine enantiomers with ­human­ (h)­ α4β2­ nicotinic­ acetylcholine receptors in different conformational states is determined by pharmacological and structural studies. The aim of this study was to show how N,6-dimethyltricyclo[5.2.1.02,6] decan-2-amine enantiomers interact with the human α4b2 nicotinic acetylcholine receptor at luminal and non-luminal domains. Materials and methods The pharmacological properties of N,6-dimethyltricyclo[5.2.1.02,6] decan-2-amine enantiomers were characterised at hα4b2 AChR by radioligand binding assays, employing the noncompetitive antagonist [3H] imipramine and the [3H]-N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine enantiomers, as well as molecular docking and dynamic studies. Results The radioligand competition results established that both enantiomers bind­with­very­low­affinity­to­the­[3H] imipramine-binding­ sites­ at­ hα4β2­ AChRs in the resting and desensitised states (apparent Ki~0.1–0.5 mM). The fact that the nH values are lower than unity (~0.5) suggests a negative cooperative interaction between imipramine and each enantiomer, and consequently that this interaction is more likely allosteric in nature or a combination of allosteric/steric interactions. The molecular docking results using the hα4b2 AChR model indicate that the (S,S)-enantiomer, in the protonated and neutral states, interact with luminal and non-luminal binding domains, whereas the (R,R)enantiomer interacts only with the intersubunit (non-luminal) locus. Interestingly, these sites are different to that for imipramine, supporting our [3H]imipramine-binding results. Discussion Our results suggest that these novel compounds do not overlap the imipramine-binding sites, but bind to a mixed luminal/non-luminal domain and to an intersubunit site formed between the α4­ and­ b2 transmembrane segments. Conclusion We hypothesise that this intersubunit interaction may impede the M2 helix rotation necessary for gating, stabilising a nonconducting conformational state. In this regard, the previously observed antidepressant activity elicited by N,6-dimethyltricyclo[5.2.1.02,6] decan-2-amine enantiomers may be produced by a novel negative allosteric modulatory mechanism.

Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional α7 AChRs.

Functional α7 nicotinic acetylcholine receptors (AChRs) do not assemble efficiently in cells transfected with α7 subunits unless the cells are also transfected with the chaperone protein RIC-3. Despite the presence of RIC-3, large amounts of these subunits remain improperly assembled. Thus, additional chaperone proteins are probably required for efficient assembly of α7 AChRs. Cholinergic ligands can act as pharmacological chaperones to promote assembly of mature AChRs and upregulate the amount of functional AChRs. In addition, we have found that the chemical chaperones 4-phenylbutyric acid (PBA) and valproic acid (VPA) greatly increase the amount of functional α7 AChRs produced in a cell line expressing both α7 and RIC-3. Increased α7 AChR expression allows assay of drug action using a membrane potential-sensitive fluorescent indicator. Both PBA and VPA also increase α7 expression in the SH-SY5Y neuroblastoma cell line that endogenously expresses α7 AChRs. VPA increases expression of endogenous α7 AChRs in hippocampal neurons but PBA does not. RIC-3 is insufficient for optimal assembly of α7 AChRs, but provides assay conditions for detecting additional chaperones. Chemical chaperones are a useful pragmatic approach to express high levels of human α7 AChRs for drug selection and characterization and possibly to increase α7 expression in vivo.

Human α4β2 Nicotinic Acetylcholine Receptor as a Novel Target of Oligomeric α-Synuclein

Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5′-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM) analyses, we find that only large (>4 nm) oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates) exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD.

Nicotinic Receptor Transduction Zone: Invariant Arginine Couples to Multiple Electron-Rich Residues

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.

Structural Analysis and Ion Translocation Mechanisms of the Muscle-Type Acetylcholine Receptor Channel

The aim of this work is to analyze the conformational changes in the acetylcholine receptor caused by channel opening and to investigate the electrostatic profile during ion translocation through the channel. A computational model of the human muscle-type acetylcholine receptor (AChR) was built and used to analyze channel structure and its interactions with different ions. Using the Torpedo AChR crystal structure as a homologous template, the 3D structure of the human muscle-type AChR was reconstructed. This first model is optimized and an open structure of the channel is generated using Normal Mode Analysis in order to assess morphologic and energetic differences between open and closed structures. In addition, the issue of ion translocation is investigated in further detail. Results elucidate different aspects of the channel: channel gate structure, channel interactions with translocating ions, differences between muscle-type AChR and previous neuronal-type AChR models. The model constructed here is ideal for further computational studies on muscle-type AChR and its pathologic mutations.

Novel 1-(1-benzyl-1H-indol-3-yl)-N,N,N-trimethylmethanaminium iodides are competitive antagonists for the human α4β2 and α7 nicotinic acetylcholine receptors

This work presents the synthesis and the pharmacological characterization of a series of novel 1-(1-benzyl-1H-indol-3-yl)-N,N,N-trimethylmethanaminium iodide derivatives at the human (h) α7 and α4β2 nicotinic acetylcholine receptors (nAChRs). The inhibitory activity of the compounds was determined by Ca2+ influx assays on cells expressing either the hα7 or hα4β2 nAChR subtype. To determine whether the observed inhibitory activity is mediated by a competitive or non-competitive mechanism, additional radioligand binding assays were performed using [3H]methyllycaconitine, [3H]cytisine, and [3H]imipramine. The results established that the compounds inhibit the nAChRs by a competitive mechanism and that the potencies are higher for the hα7 nAChR compared to that for the hα4β2 nAChR. Substitutions with oxygenated functional groups on the benzene ring increase the receptor selectivity. In particular, the hydroxyl derivatives 4b and 4c present the highest selectivity for the hα7 nAChR subtype. Molecular docking results indicate that the hydroxyl group forms a hydrogen bond with the carbonyl group at α7-Gln116, but not at β2-Phe115, supporting the observed receptor selectivity at the molecular level.

Myasthenogenicity of the main immunogenic region

In myasthenia gravis (MG) and experimental autoimmune MG (EAMG), many pathologically significant autoantibodies are directed to the main immunogenic region (MIR) of muscle nicotinic acetylcholine receptors (AChRs), a conformation-dependent region at the extracellular tip of α1 subunits of AChRs. Human muscle AChR α1 MIR sequences were integrated into Aplesia ACh-binding protein (AChBP). The chimera potently induced EAMG, while AChBP induced EAMG much less potently. AChBP is a water-soluble protein resembling the extracellular domain of AChRs; yet, rats immunized with chimeras developed autoantibodies to both extracellular and cytoplasmic domains of muscle AChRs. We propose that an initial autoimmune response directed at the MIR leads to an autoimmune response sustained by muscle AChRs. Autoimmune stimulation sustained by endogenous muscle AChR may be a target for specific immunosuppression. These studies show that the α1 MIR is highly myasthenogenic, and that AChR-like proteins distantly related to muscle AChR can induce EAMG and, potentially, MG.

Tobacco Nitrosamine N-nitrosonornicotine as Inhibitor of Neuronal Nicotinic Acetylcholine Receptors

Nitrosamines are well known for their carcinogenic potential. Recently, it was found that some of them may also interact with human nicotinic acetylcholine receptor (nAChR) subtypes. This work studied the effects of N-nitrosonornicotine (NNN) on recombinant rat α3β4 nAChR in HEK cells as well as on nAChR endogenously expressed in PC12 pheochromocytoma cells and in BC3H1 muscle-type cells. Whole-cell recording in combination with the cell-flow technique for agonist and inhibitor application in the millisecond time region revealed that NNN inhibits the activity of neuronal nAChR expressed in HEK or PC12, whereas weak inhibitory effects on muscle-type nAChR were observed at NNN concentrations up to 3mM. Pharmacological actions of NNN and the inhibition mechanism were studied in detail using recombinant α3β4 nAChR expressed in HEK cells as a model. NNN-induced inhibition of nicotine-evoked α3β4 nAChR activity was dose-dependent with an inhibitory constant (IC(50)) of 0.92 ± 0.05mM. Analysis based on mathematical models indicated a noncompetitive inhibition mechanism of the rat α3β4 nAChR by NNN. NNN's mechanism of action involves acceleration of conversion of the receptor from active to desensitized forms. In summary, this work shows that NNN inhibits rat α3β4 nAChR in a noncompetitive way and interacts weakly with muscular nAChR.

Peptide mimics of the torpedo and human AChR main immunogenic regions

Peptide mimics of the torpedo and human AChR main immunogenic regions