Human Acetylcholine Research Articles

Vibrational spectroscopy analysis of ligand efficacy in human M2 muscarinic acetylcholine receptor (M2R)

The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.

An in vitro model to study the human specific nicotinic acetylcholine receptor gene CHRFAM7A

An in vitro model to study the human specific nicotinic acetylcholine receptor gene CHRFAM7A

Effects of C-Terminal Carboxylation on α-Conotoxin LsIA Interactions with Human α7 Nicotinic Acetylcholine Receptor: Molecular Simulation Studies

α-Conotoxins selectively bind to nicotinic acetylcholine receptors (nAChRs), which are therapeutic targets due to their important role in signaling transmission in excitable cells. A previous experimental study has demonstrated that carboxylation of the C-terminal of α-conotoxin LsIA reduces its potency to inhibit human α7 nAChR relative to naturally amidated LsIA. However, little is known about the contribution of conformational changes in the receptor and interactions, induced by C-terminal amidation/carboxylation of conotoxins, to selective binding to nAChRs, since most conotoxins and some disulfide-rich peptides from other conotoxin subfamilies possess a naturally amidated C-terminal. In this study, we employ homology modeling and molecular dynamics (MD) simulations to propose the determinants for differential interactions between amidated and carboxylated LsIAs with α7 nAChR. Our findings indicate an overall increased number of contacts favored by binding of amidated LsIA versus its carboxylated counterpart. Toxin-receptor pairwise interactions, which may play a role in enhancing the potency of the former, include ARG10-TRP77, LEU141 and CYS17-GLN79 via persistent hydrogen bonds and cation-π interactions, which are weakened in the carboxylated form due to a strong intramolecular salt-bridge formed by ARG10 and carboxylated C-terminus. The binding of amidated LsIA also induces enhanced movements in loop C and the juxtamembrane Cys-loop that are closely associated with receptor function. Additionally, the impacts of binding of LsIA on the overall structure and inter-subunit contacts were examined using inter-residue network analysis, suggesting a clockwise tilting of the α7 C and F loops upon binding to carboxylated LsIA, which is absent for amidated LsIA binding. The predicted molecular mechanism of LsIA binding to the α7 receptor may provide new insights into the important role of the C-terminal in the binding potency of conotoxins at neuronal nAChRs for pharmacological purposes.

Molecular interactions of type I and type II positive allosteric modulators with the human α7 nicotinic acetylcholine receptor: an in silico study

The binding site locations and structural components for type I and type II positive allosteric modulators (PAMs) of the α7 nicotinic acetylcholine receptor (nAChR) have not been fully characterized yet. In this regard, homology models of the human α7 nAChR and hα7/m5-HT3A chimera, built using the crystal structure of the serotonin type 3A receptor (5-ΗΤ3ΑR), were used for molecular docking and molecular dynamics simulations to study the molecular interactions of selected type I (5-hydroxyindol, NS-1738, and LY-2087101) and type II (PNU-120596, PAM-2, and TBS-516) PAMs. The docking results indicate: (1) a site located in the extracellular domain (ECD) for type I PAMs such as NS-1738 and LY-2087101, but not for 5-HI; (2) an overlapping site in the ECD–transmembrane domain (TMD) junction for all studied PAMs. Additional docking results on the hα7/m5-HT3A chimera supported experimental results indicating that the ECD site might be relevant for type I PAM activity; and (3) two TMD sites, an intrasubunit site that recognizes type II PAMs, and an intersubunit pocket with high specificity for 5-HI (type I PAM). The in silico α7TSLMF mutant results support the view that M1–Ser223 and M3–Ile281 are key residues for the interaction of PAM-2 and PNU-120596 with the intrasubunit cavity. Our in silico results are in agreement with experimental data showing that the intrasubunit cavity is relevant for the activity of type II PAMs, and suggest that the ECD–TMD junction and intersubunit sites could be significant for the activity of type I PAMs.

Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant

Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

Cellular Transplantation as the Treatment of Alzheimer’s Diseas e in Mouse Models

Acetylcholine (Ach) and N-methyl-D-aspartate (NMDA) have been two major therapeutic targets of Alzheimer’s disease (AD) for decade. However, truly effective remedy for AD has not been successfully developed. We previously transplanted neurons derived from human induced pluripotent stem (hiPS) cells into the hippocampus of human amyloid precursor protein transgenic AD model mice. The cell transplantation significantly improved cognitive dysfunction in the dementia model mice. Human choline acetyl transferase (ChAT) positive cholinergic neurons located throughout the cortex of the grafted mice. Human and mouse ChAT positive neurons and alpha7 nicotinic acetylcholine receptor (α7nAChR) positive neurons significantly increased in the cortex and hippocampus of the grafted dementia mice compared with the vehicle injected dementia mice. Human and mouse vesicular GABA transporter (VGAT) positive neurons distributed mainly in the hippocampus and, though the number was small, human VGAT positive neurons located in the cortex. In the grafted mouse cortex, the number of GABA receptor (GABAR) positive neurons of both hiPS origin and mouse origin increased significantly compared with those in the vehicle injected mouse cortex. We suggested that positive feedback loops of neurotransmitter secretion of the cortex and hippocampus induced the characteristic distribution of the transplanted neurons. In this review, we summarized current advances in stem cell therapy for dementia model mice, especially to highlight the relationships between major neurotransmitters and host/transplanted neurons.

Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.

Regulation of the “neuronal” nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

Aspartame - Induced Toxicity on Human Erythrocyte Membrane Acetylcholine Esterase Activity

Aspartame - Induced Toxicity on Human Erythrocyte Membrane Acetylcholine Esterase Activity

The Protective Role of Vitamin E and L Carnitine on Human Erythrocyte Membrane Acetylcholine Esterase Activity

The Protective Role of Vitamin E and L Carnitine on Human Erythrocyte Membrane Acetylcholine Esterase Activity

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.

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.

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

Antipsychotic compounds show inhibition of the human nicotinic acetylcholine α-7 receptor at high concentrations

Antipsychotic compounds show inhibition of the human nicotinic acetylcholine α-7 receptor at high concentrations

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.

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.

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.

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.

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.

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.