Human Alpha7 Nicotinic Acetylcholine Receptor Research Articles

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of five homologous subunits. The homopentameric α7-nAChR, abundantly expressed in the brain, is involved in the regulation of the neuronal plasticity and memory and undergoes phosphorylation by protein kinase A (PKA). Here, we extracted native α7-nAChR from murine brain, validated its assembly by cryo-EM and showed that phosphorylation by PKA in vitro enables its interaction with the abundant human brain protein 14-3-3ζ. Bioinformatic analysis narrowed the putative 14-3-3-binding site down to the fragment of the intracellular loop (ICL) containing Ser365 (Q361RRCSLASVEMS372), known to be phosphorylated in vivo. We reconstructed the 14-3-3ζ/ICL peptide complex and determined its structure by X-ray crystallography, which confirmed the Ser365 phosphorylation-dependent canonical recognition of the ICL by 14-3-3. A common mechanism of nAChRs’ regulation by ICL phosphorylation and 14-3-3 binding that potentially affects nAChR activity, stoichiometry, and surface expression is suggested.

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1–10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1–10 mg/kg, sc) and set shift performance in rats (1–10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1–3 mg/kg, po). BMS-933043 also improved auditory gating (0.56–3 mg/kg, sc) and mismatch negativity (0.03–3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans.

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.

Cellular and molecular mechanisms of the restoration of human APP transgenic mouse cognitive dysfunction after transplant of human iPS cell-derived neural cells

Cholinergic neuronal loss is a common finding in patients with Alzheimer's disease (AD) and AD model mice. We previously transplanted neurons derived from human induced pluripotent stem (iPS) cells into the hippocampus of human amyloid precursor protein transgenic AD model mice. In the present study, we examined the cellular and molecular mechanisms involved in the alleviation of cognitive dysfunction in transplanted mice.After transplant, mice showed improvement in cognitive function, confirming our previous findings. Human choline acetyltransferase (ChAT)-positive cholinergic neurons were distributed throughout the cortex of the grafted mice. Human and mouse ChAT-positive neurons and alpha7 nicotinic acetylcholine receptor (α7nAChR)-positive neurons were significantly increased in the cortex and hippocampus of the grafted mice compared with the vehicle-injected mice. In addition, human and mouse vesicular GABA transporter (VGAT)-positive neurons were located mainly in the hippocampus and, though the number was small, human VGAT-positive neurons were observed in the cortex. In the grafted mouse cortex, the number of GABA receptor (GABAR)-positive neurons of both human origin and mouse origin were significantly increased compared with those in the vehicle-injected mouse cortex. The α7nAChR-positive and GABAR-positive neurons expressed phosphorylated Akt and c-fos in the cortex, suggesting that these receptor-expressing neurons were possibly activated by the neurotransmitters secreted from the grafted neurons.Collectively, the grafted and host neurons may form positive feedback loops via neurotransmitter secretion in both the cerebral cortex and hippocampus, leading to alleviation of cognitive dysfunction in dementia model mice.

Potential implications of human alpha7 nicotinic acetylcholine receptor genetic variants to the pathogenesis and treatment of HIV‐associated neurocognitive disorders

The α7 nicotinic acetylcholine receptor (nAChR), a ligand gated ion channel characterized by its high calcium permeability, was shown to be upregulated in SH‐SY5Y cells exposed to HIV‐1 glycoprotein gp120 leading to Ca2+ overloading and cell death. These results suggest that HIV‐associated neurocognitive disorders (HAND) could be related to increased neuronal death as result of excessive Ca2+ influx through the upregulated α7 nAChR. Furthermore, these results also suggest that α7 nAChR antagonists can be exploited for neuroprotection in HIV‐1 seropositive patients. Genetic variants of the CHRNA7 gene (encodes α7 nAChR) that result in ion channels with increased expression and/or functionality could thus underlie increased risk of HAND. In addition, CHRNA7 genetic variants could also result in receptors with diminished response to antagonists. The hypothesis is that non‐synonymous single nucleotide polymorphisms (SNPs) located in the CHRNA7 gene could result in α7 nAChRs with enhanced functionality and/or expression levels, and with altered responses to drugs aimed at interfering with the excessive Ca2+ flow. Bupropion was studied as a model α7 nAChR antagonist. Herein we present data demonstrating that genetic variants of the CHRNA7 gene may result in α7 nAChRs with altered functionality and response to bupropion. Supported by NIMH grant # P30MH075673–07 and NIMHD grant # 8U54MD007587–03.

Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper

A large number of structurally diverse ligands have been produced to selectively target α7 nicotinic acetylcholine receptors (nAChRs). We applied the method of scanning cysteine accessibility mutations (SCAM) to the ligand-binding domain of the α7 nAChR to identify subdomains of particular importance to the binding and subsequent activation by select agonists. We evaluated the activity of four structurally distinct α7 agonists on wild-type human α7 and 44 targeted mutants expressed in Xenopus oocytes. Responses were measured prior and subsequent to the application of the sulfhydryl reagent methanethiosulfonate ethylammonium (MTSEA). One mutant (C116S) served as a Cys-null control, and the additional mutants were made in the C116S background. In many cases, the insertion of free cysteines into the agonist-binding site had a negative effect on function, with 12 of 44 mutants showing no detectable responses to ACh, and with only 19 of the 44 mutants showing sufficiently large responses to permit further study. Several of the cysteine mutations, including W55C, showed selectively reduced responses to the largest agonist tested, 2-methoxy,4-hydroxy-benzylidene anabaseine. Interestingly, although homology models suggest that most of the introduced cysteine mutations should have had good solvent accessibility, application of MTSEA had no effect or produced only modest changes in the agonist response profile of most mutants. Consistent with previous studies implicating W55 to play important roles in agonist activation, MTSEA treatment further decreased the functional responses of W55C to all the test agonists. While the cysteine mutation at L119 itself had relatively little effect on receptor function, treatment of L119C receptors with MTSEA or alternative cationic sulfhydryl reagents profoundly decreased activation by all agonists tested, suggesting a general block of gating. The homologous mutation in heteromeric nAChRs produced similar results, provided that the mutation was placed in the beta subunit complementary surface of the ligand-binding domain. Structural models locate the L119 residue directly across the subunit interface from the C-loop of the primary face of the binding domain. Our data suggest that a covalent modification of L119C by MTSEA or other cationic reagents might block the binding of even small agonists such as TMA through electrostatic interactions. Reaction of L119C with small non-polar reagents increases activation by small agonists but can block the access of large ligands such as benzylidene anabaseines to the ligand-binding domain.

Interaction of Selective Serotonin Reuptake Inhibitors with Neuronal Nicotinic Acetylcholine Receptors

We compared the interaction of fluoxetine and paroxetine, two selective serotonin reuptake inhibitors (SSRIs), with the human (h) alpha4beta2, alpha3beta4, and alpha7 nicotinic acetylcholine receptors (AChRs) in different conformational states, using Ca(2+) influx, radioligand binding, and molecular docking approaches. The results established that (1) fluoxetine was more potent than paroxetine in inhibiting agonist-activated Ca(2+) influx on halpha4beta2 and halpha7 AChRs, whereas the potency of both SSRIs was practically the same in the halpha3beta4 AChR. [corrected] (2) SSRIs bind to the [(3)H]imipramine locus with a [corrected] higher affinity when the AChRs are in the desensitized states compared to the resting states. (3) The different receptor specificity for fluoxetine determined by their inhibitory potencies or binding affinities suggests different modes of interaction when the AChR is in the closed or activated state. (4) Neutral and protonated fluoxetine interacts with a binding domain located in the middle of the AChR ion channel. In conclusion, SSRIs inhibit the most important neuronal AChRs with potencies and affinities that are clinically relevant by binding to a luminal site that is shared with tricyclic antidepressants.

Different Interaction between the Agonist JN403 and the Competitive Antagonist Methyllycaconitine with the Human α7 Nicotinic Acetylcholine Receptor

The interaction of the agonist JN403 with the human (h) alpha7 nicotinic acetylcholine receptor (AChR) was compared to that for the competitive antagonist methyllycaconitine (MLA). The receptor selectivity of JN403 was studied on the halpha7, halpha3beta4, and halpha4beta2 AChRs. The results established that the cationic center and the hydrophobic group found in JN430 and MLA are important for the interaction with the AChRs. MLA preincubation inhibits JN403-induced Ca(2+) influx in GH3-halpha7 cells with a potency 160-fold higher than that when MLA is co-injected with JN403. The most probable explanation, based on our dynamics results, is that MLA (more specifically the 3-methyl-2,5-dioxopyrrole ring and the B-D rings) stabilizes the resting conformational state. The order of receptor specificity for JN403 is as follows: halpha7 > halpha3beta4 ( approximately 40-fold) > halpha4beta2 ( approximately 500-fold). This specificity is based on a larger number of hydrogen bonds between the carbamate group (another pharmacophore) of JN403 and the halpha7 sites, the electrostatic repulsion between the positively charged residues around the halpha3beta4 sites and the cationic center of JN403, fewer hydrogen bonds for the interaction of JN403 with the halpha3beta4 AChR, and an unfavorable van der Waals interaction between JN403 and the alpha4-beta2 interface. The higher receptor specificity for JN403 could be important for the treatment of alpha7-related disorders, including dementias, pain-related ailments, depression, anxiety, and wound healing.

Potentials of Mean Force for Acetylcholine Unbinding from the Alpha7 Nicotinic Acetylcholine Receptor Ligand-Binding Domain

The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here, we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.

Unexpected sensitivity of the human α7 neuronal nicotinic acetylcholine receptor to aminoglycosides

The wide use of antibiotics and the development of resistance is a major health concern and, despite their relatively severe side effects, aminoglycoside antibiotics are still used in clinics. Effects of seven aminoglycosides were investigated at the human homomeric alpha7 and heteromeric alpha4beta2 neuronal nicotinic acetylcholine receptors. All aminoglycosides tested inhibited the acetylcholine-evoked responses with more pronounced effects at alpha7 than at alpha4beta2. Neomycin displayed higher blockade with a half inhibition in the nanomolar range at low calcium concentration and in the micromolar range in physiological calcium concentration but still exerted blockade below the concentration used in the clinic. These data suggest that some of their side effects may be attributable to their interactions with neuronal nicotinic acetylcholine receptors.

α7-Acetylcholine receptor antibodies in two patients with Rasmussen encephalitis

Rasmussen encephalitis (RE) sera were screened for antibodies to human alpha7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through alpha7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited (125)I-alpha-bungarotoxin binding in cells expressing alpha7 nAChRs. Thus, the alpha7 nAChR is a potential target for pathogenic antibodies in patients with RE.

Further evidence for association of promoter variants in human alpha7 nicotinic acetylcholine receptor subunit gene (CHRNA7) with schizophrenia

Further evidence for association of promoter variants in human alpha7 nicotinic acetylcholine receptor subunit gene (CHRNA7) with schizophrenia

Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human alpha 7-nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) containing alpha7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional alpha7-nAChR has only been achieved in a few host cell types, such as the SH-EP1 human epithelial cell line. All ionotropic glycine receptor, GABA(A) receptor, 5-HT(3) receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for alpha7 subunits) in their N-terminal extracellular domain. These residues are thought to be involved in the formation of a conserved cystine loop that is critical to the proper folding and assembly of subunits. However, nAChR alpha7 (and alpha8) subunits also contain a third cysteine residue, C138, N-terminal to the conserved cysteine pair. Using SH-EP1 cells as a host for heterologous expression, we evaluated the roles of C138, C150, and C164 in subunit folding, assembly, and cell surface expression and function of alpha7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form (125)I-labeled alpha-bungarotoxin binding sites expressed on the cell surface. Further, whole-cell patch clamp recordings demonstrate that mutation of C138 to alanine does not alter the function of the fully assembled alpha7-nAChR. These results indicate that C150 and C164 are required for surface expression, but that C138 is neither necessary for nor inhibitory toward the surface expression and function of human alpha7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on alpha7-nAChR assembly or function.

Effects of the plant alkaloid tetrandrine on human nicotinic acetylcholine receptors.

Functional effects of the well-characterized antagonist of L-type Ca(2+) channels tetrandrine on recombinant human gamma-aminobutyric acid type A (GABA(A)) (alpha1beta2gamma2s) receptor or human alpha7, alpha4beta2, alpha1beta1deltagamma and alpha1beta1delta epsilon nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage clamp. Tetrandrine inhibited the function of acetylcholine nicotinic receptors, but it had no effect on GABA(A) receptors. Potency of inhibition was influenced by the receptor subtype and the rank order was alpha4beta2>alpha7>alpha1beta1deltagamma congruent with alpha1beta1delta epsilon. Functional inhibition of alpha4beta2 and alpha1beta1deltagamma receptors was noncompetitive, but only inhibition of alpha1beta1deltagamma receptors was voltage-dependent. Binding of 125I-alpha-bungarotoxin to alpha1beta1deltagamma or 3H-cytisine to alpha4beta2 receptors was also inhibited by tetrandrine, but inhibition was noncompetitive and required concentrations higher than those needed to inhibit receptor function. Inhibition of both alpha7 receptor function and binding of 125I-alpha-bungarotoxin to alpha7 receptor were mixed competitive/noncompetitive and occurred at a similar concentration range.

Ivermectin: a positive allosteric effector of the alpha7 neuronal nicotinic acetylcholine receptor.

We report that preapplication of ivermectin, in the micromolar range, strongly enhances the subsequent acetylcholine-evoked current of the neuronal chick or human alpha7 nicotinic acetylcholine receptors reconstituted in Xenopus laevis oocytes and K-28 cells. This potentiation does not result from nonspecific Cl- currents. The concomitant increase in apparent affinity and cooperativity of the dose-response curve suggest that ivermectin acts as a positive allosteric effector. This interpretation is supported by the observation of an increase in efficiency of a partial agonist associated with the potentiation and by the differential effect of ivermectin on mutants within the M2 channel domain. Ivermectin effects reveal a novel allosteric site for pharmacological agents on neuronal alpha7 nicotinic acetylcholine receptors.