Human Neuronal Nicotinic Acetylcholine Receptors Research Articles

Up-regulation of human alpha7 nicotinic receptors by chronic treatment with activator and antagonist ligands.

This study examined the binding and functional properties of human alpha7 neuronal nicotinic acetylcholine receptors stably expressed in human embryonic kidney (HEK) 293 cells following chronic treatment with nicotinic receptor ligands. Treatment of cells with (-)-nicotine (100 microM) for 120 h increased the Bmax values of [125I]alpha-bungarotoxin binding 2.5-fold over untreated cells. This effect was concentration-dependent (EC50) = 970 microM) and a 6-fold upregulation was observed with the maximal concentration of (-)-nicotine tested. Also, treatment of cells with ligands of varying intrinsic activities including (+/-)-epibatidine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) also upregulated [125I]alpha-bungarotoxin binding. A concentration-dependent upregulation of binding sites was also observed following treatment with the alpha7 nicotinic receptor antagonist, methyllycaconitine (EC50 = 92 microM) with a maximal upregulation of about 7-fold. Functionally, the peak amplitude of the whole-cell currents recorded by fast application of (-)-nicotine after chronic treatment of cells with concentrations of (-)-nicotine (1000 microM) or methyllycaconitine (10 microM) that elicited similar increases in binding levels (3.5-fold) resulted in increases of 2-fold (505 +/- 21 pA) and 6-fold (1820 +/- 137 pA) respectively in whole cell current amplitude compared to untreated cells (267 +/- 24 pA). These studies clearly demonstrate that long-term exposure to both activator and antagonist ligands can increase the density of alpha7 nicotinic receptors and can differentially enhance nicotinic receptor function.

Open-channel blockers at the human alpha4beta2 neuronal nicotinic acetylcholine receptor.

To extend our knowledge of the pharmacological profile of human alpha4beta2 neuronal nicotinic receptors, we investigated the action of hexamethonium on the major brain human nicotinic acetylcholine receptor (nAChR) stably expressed in human embryonic kidney 293 cells. This compound displays all of the characteristics of an open-channel blocker at the human alpha4beta2 nAChR: a voltage-dependent inhibition (more pronounced at hyperpolarized potentials), absence of competition, and use dependence. Moreover, we observed that classic N-methyl-D-aspartate open-channel blockers amantadine, 3,5-dimethyl-1-adamantanamine (memantine), and dizocilpine [(+)-MK-801] and the calcium channel antagonist 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate are powerful inhibitors of the human alpha4beta2 nAChR. Dose-inhibition curves yield, at -100 mV, IC50 values in the micromolar range for all of compounds and Hill coefficients below unity. Whole-cell current-voltage relationships display a strong rectification profile at hyperpolarized potentials, and current blockades are fitted adequately by a mathematical model that describes the mechanism of an ion channel block. We conclude that these molecules are powerful human alpha4beta2 open-channel blockers ranking in the following order of potency: amantadine > memantine = hexamethonium > 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate approximately (+)-MK-801.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than non-specific agents such as nicotine.

Regulation of Human α4β2 Neuronal Nicotinic Acetylcholine Receptors by Cholinergic Channel Ligands and Second Messenger Pathways

The alpha4beta2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human alpha4beta2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the Ki values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)-nicotine or dihydro-beta-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.

Cloning and sequence of full-length cDNAs encoding the human neuronal nicotinic acetylcholine receptor (nAChR) subunits β3 and β4 and expression of seven nAChR subunits in the human neuroblastoma cell line SH-SY5Y and/or IMR-32

Using PCR-based techniques, we have isolated and sequenced the full-length cDNAs that encode the human neuronal nAChR subunits α3,4,5,7 and β2,3,4 in the neuroblastoma cell lines SH-SY5Y and/or IMR-32. The predicted nAChR β3- and β4-subunit proteins contain 458 and 498 amino acids, respectively. Except for the β2, all the other cloned cDNAs showed differences with the published sequences. Northern blots show expression of the nAChR subunits α3,7 and β2,4 in the human neuroblastoma cell line SH-SY5Y, with intensity of the hybridisation signal decreasing in the order α3>β4>β2>α7.

Human α4β2 Neuronal Nicotinic Acetylcholine Receptor in HEK 293 Cells: A Patch-Clamp Study

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, alpha4 and beta2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 microM and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human alpha4beta2 receptors is (-)-nicotine > ACh > (-)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 >> (-)-nicotine > ACh >> (-)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 microM ACh with known nAChR inhibitors such as dihydro-beta-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50 values of 80 nM and 1.5 microM. The current-voltage relationship of human alpha4beta2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human alpha4beta2 nAChRs are permeable to sodium and potassium ions. In the "outside-out" configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.

Short Report on DNA Marker at Candidate Locus: Dinucleotide polymorphism in the first intron of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

Short Report on DNA Marker at Candidate Locus: Dinucleotide polymorphism in the first intron of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

Exon–Intron Structure of the Human Neuronal Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4)

The human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idiopathic epilepsies.

Detection of a CfoI polymorphism within exon 5 of the human neuronal nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4).

The gene coding for the alpha 4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently mapped in the candidate region for benign familial neonatal convulsions (BFNC) on chromosome 20q13.2-q13.3. The region is only partially covered with polymorphic markers, and so far no PCR-based polymorphisms have been described in the critical region for BFNC. We now report the first polymorphic marker in the coding region of CHRNA4. The new marker, which is detected by PCR, will be useful for evaluation of the role of CHRNA4 as a candidate gene for BFNC. It will further enable the investigation of this important brain-specific gene in association studies with different types of epileptic diseases and other neurological disorders.

Cloning and transient expression of genes encoding the human α4 and β2 neuronal nicotinic acetylcholine receptor (nAChR) subunits

Partial cDNA clones generated by RT-PCR were used as probes to clone the cDNAs encoding the human α4 and β2 neuronal nicotinic acetylcholine receptor (nAChR) subunits. The 2.1-kb α4 cDNA shows 84 and 76% identity to the rat and chicken cDNA sequences, respectively. The deduced amino-acid sequence shares 89 and 84% similarity, respectively, with the corresponding rat and chicken proteins, with most of the divergence occurring in the cytoplasmic domain. The 1721-nucleotide β sequence was identical to the human β2 sequence previously reported. Transfection of the α4 and β2 clones into HEK293 cells resulted in the formation of binding sites that display high affinity towards [ 3h]cytisine, a characteristic of the α4β2 subtype produced in vivo

Molecular cloning of a human neuronal nicotinic acetylcholine receptor β3-like subunit

Three cDNA clones homologous to rat neuronal nicotinic receptors were identified in a human brain stem library by screening at low stringency with a mixture of rat α5, β2 and β4 subunit probes. Positive clones were further analysed by hybridisation at higher stringency with individual rat nicotinic receptor probes. One positive clone was found to encode a protein that exhibited characteristic features of a member of the ligand-gated ion channel class of protein subunits, and showed 89% homology with a rat β3 neuronal nicotinic receptor subunit. Northern blots demonstrated the presence of a 1.7 kb transcript in RNA extracted from adult human Pons. This clone has therefore been designated the human β3 neuronal nicotinic receptor subunit.

Chromosomal localization of seven neuronal nicotinic acetylcholine receptor subunit genes in humans

We have determined the chromosomal location of seven human neuronal nicotinic acetylcholine receptor subunit genes by genomic Southern analysis of hamster/human somatic cell hybrid DNAs. The β2 subunit gene was localized to human chromosome 1, the α2 and β3 subunit genes were localized to human chromosome 8, the α3, α5, and β4 subunit genes were localized to human chromosome 15, and the α4 subunit gene was localized to human chromosome 20. Mapping of the β2 subunit gene to chromosome 1 establishes a syntenic group with the amylase gene locus on human chromosome 1 and mouse chromosome 3, while mapping of the α3 subunit gene to chromosome 15 confirms the existence of a syntenic group with the mannose phosphate isomerase gene locus on human chromosome 15 and mouse chromosome 9.

Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies

Neuronal acetylcholine receptors (AChRs), which bind nicotine with high affinity but do not bind alpha-bungarotoxin (alpha Bgt), have recently been immunoaffinity-purified from chicken (Whiting and Lindstrom, 1986a) and rat (Whiting and Lindstrom, 1987a) brain using monoclonal antibodies (mAbs). Here we report the characterization of nicotinic AChRs of bovine and human brain using as probes mAbs prepared to AChRs from rat brain. Both the human and bovine brain AChRs exhibit high affinity for L-nicotine (Ki = 16 nM for bovine AChR and Ki = 6.5 nM for human AChR) and other cholinergic agonists, relatively lower affinity for cholinergic antagonists, and do not bind alpha Bgt. These AChRs are affinity-labeled with bromoacetylcholine and 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA) after reduction with dithiothreitol, indicating that amino acid residues homologous to cysteines 192 and 193 of alpha subunits of Torpedo electric organ AChRs are conserved. Immunoaffinity-purified bovine brain AChR consists of 2 types of subunit, Mr 50,600 and Mr 74,400. The Mr 74,400 subunit was affinity-labeled with 3H-MBTA, indicating that it contains the ACh binding site. Thus, mAbs have proven to be excellent probes for these proteins, and have been used to show that neuronal nicotinic AChRs of chickens, rats, and cattle are macromolecules approximately 10 S in size and composed of only 2 kinds of subunits: an ACh-binding subunit and a structural subunit.