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1.
Mol Pharmacol ; 98(2): 168-180, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32474444

RESUMO

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4ß2 and α7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 1'-N-methyl with an ethyl group or adding a second 1'-N-methyl group significantly reduced interaction with α4ß2 but not α7 receptors. The 2'-methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3'- and 5'-trans-methylations were much better tolerated by α7 receptors than α4ß2 receptors, 4'-methylation decreased potency and efficacy at α7 receptors much more than at α4ß2 receptors. Whereas cis-5'-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-5'-methylnicotine retained considerable α7 receptor activity. Differences between the two 5'-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 5'-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 1'-, 3'-, and 5'-methylations and the greater effects of 2'- and 4'-methylations on nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold. SIGNIFICANCE STATEMENT: Using a comprehensive "methyl scan" approach, we show that the orthosteric binding sites for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact differently with the pyrrolidinium ring of nicotine, and we suggest reasons for the higher affinity of nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were identified that may be useful in the design of new drugs targeting these receptors.


Assuntos
Nicotina/análogos & derivados , Piridinas/síntese química , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Sítios de Ligação , Masculino , Metilação , Simulação de Acoplamento Molecular , Estrutura Molecular , Nicotina/química , Piridinas/química , Piridinas/farmacologia , Ratos , Relação Estrutura-Atividade , Xenopus laevis
2.
Neuropharmacology ; 168: 108021, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32146229

RESUMO

Postsynaptic nAChRs in the peripheral nervous system are critical for neuromuscular and autonomic neurotransmission. Pre- and peri-synaptic nAChRs in the brain modulate neurotransmission and are responsible for the addictive effects of nicotine. Subtypes of nAChRs in lymphocytes and non-synaptic locations may modulate inflammation and other cellular functions. All AChRs that function as ligand-gated ion channels are formed from five homologous subunits organized to form a central cation channel whose opening is regulated by ACh bound at extracellular subunit interfaces. nAChR subtype subunit composition can range from α7 homomers to α4ß2α6ß2ß3 heteromers. Subtypes differ in affinities for ACh and other agonists like nicotine and in efficiencies with which their channels are opened and desensitized. Subtypes also differ in affinities for antagonists and for positive and negative allosteric modulators. Some agonists are "silent" with respect to channel opening, and AChRs may be able to signal metabotropic pathways by releasing G-proteins independent of channel opening. Electrophysiological studies that can resolve single-channel openings and molecular genetic approaches have allowed characterization of the structures of ligand binding sites, the cation channel, and the linkages between them, as well as the organization of AChR subunits and their contributions to function. Crystallography and cryo-electron-microscopy are providing increasing insights into the structures and functions of AChRs. However, much remains to be learned about both AChR structure and function, the in vivo functional roles of some AChR subtypes, and the development of better pharmacological tools directed at AChRs to treat addiction, pain, inflammation, and other medically important issues. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.


Assuntos
Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Ligantes , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/uso terapêutico , Antagonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/uso terapêutico , Dor/tratamento farmacológico , Dor/metabolismo , Estrutura Secundária de Proteína , Receptores Nicotínicos/química , Transdução de Sinais/efeitos dos fármacos
3.
Front Immunol ; 8: 603, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28620373

RESUMO

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves' disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.

4.
FASEB J ; 31(4): 1398-1420, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28100642

RESUMO

This study investigates-for the first time to our knowledge-the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3- and ß4-subunit-containing human nicotinic acetylcholine receptors (α3ß4*-nAChRs). Concatenated gene constructs were used to express precisely defined α3ß4*-nAChR isoforms (α3ß4)2ß4-, (α3ß4)2α3-, (α3ß4)2α5(398D)-, and (α3ß4)2α5(398N)-nAChR in Xenopus oocytes. In the presence or absence of lynx1, α3ß4*-nAChR agonist responses were recorded by using 2-electrode voltage clamp and single-channel electrophysiology, whereas radioimmunolabeling measured cell-surface expression. Lynx1 reduced (α3ß4)2ß4-nAChR function principally by lowering cell-surface expression, whereas single-channel effects were primarily responsible for reducing (α3ß4)2α3-nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3-fold reduction in the proportion of long bursts), and enhanced closed dwell times (3- to 6-fold increase)]. Alterations in both cell-surface expression and single-channel properties accounted for the reduction in (α3ß4)2α5-nAChR function that was mediated by lynx1. No effects were observed when α3ß4*-nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3ß4*-α5*-nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(-)-interface containing] by which prototoxins can modulate nAChR function.-George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform-specific mechanisms of α3ß4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Receptores Nicotínicos/metabolismo , Potenciais de Ação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Proteínas Ligadas por GPI/genética , Humanos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Xenopus
5.
eNeuro ; 3(4)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27559543

RESUMO

Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the ß2 subunit (ß2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the ß2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Proteoma/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Animais , Córtex Cerebral/patologia , Cotinina/metabolismo , Feminino , Humanos , Transtornos Mentais/metabolismo , Transtornos Mentais/patologia , Camundongos Transgênicos , Receptores Nicotínicos/genética , Fumar/metabolismo , Fumar/patologia , Tabagismo/metabolismo , Tabagismo/patologia
6.
Neuropharmacology ; 102: 158-73, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26561946

RESUMO

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and ß2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for ß2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and ß2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4ß2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(ß2)3] or low-sensitivity [LS; (α4)3(ß2)2] via 1:10 or 30:1 [α4:ß2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:ß2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). ß2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4ß2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using ß2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the ß2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.


Assuntos
Epilepsia do Lobo Frontal/genética , Receptores Nicotínicos/genética , Animais , Mutação , Oócitos , Xenopus
7.
J Biol Chem ; 291(5): 2444-59, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26644472

RESUMO

Two α4ß2 nicotinic acetylcholine receptor (α4ß2-nAChR) isoforms exist with (α4)2(ß2)3 and (α4)3(ß2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)ß2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)ß2 sites. However, the relative roles of the conserved α4(+)/(-)ß2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4ß2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of ß2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (ß2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)ß2 sites contribute equally to function, making identical ß2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)ß2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.


Assuntos
Regulação da Expressão Gênica , Receptores Nicotínicos/química , Acetilcolina/química , Sítio Alostérico , Animais , Azetidinas/química , Sítios de Ligação/genética , DNA Complementar/metabolismo , Eletrofisiologia , Humanos , Mutagênese Sítio-Dirigida , Mutação , Nicotina/química , Agonistas Nicotínicos/química , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Isoformas de Proteínas , RNA/metabolismo , Xenopus laevis
8.
Elife ; 3: e01473, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24497543

RESUMO

In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light-dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep. DOI: http://dx.doi.org/10.7554/eLife.01473.001.


Assuntos
Proteínas de Drosophila/metabolismo , Receptores Nicotínicos/metabolismo , Sono , Animais , Ritmo Circadiano , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulação da Expressão Gênica , Genótipo , Homeostase , Mutação , Fenótipo , Fotoperíodo , Receptores Nicotínicos/genética , Transdução de Sinais , Sono/genética , Fatores de Tempo
9.
J Pharmacol Exp Ther ; 348(1): 46-58, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24190916

RESUMO

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(ß2)3) or low-sensitivity (LS) (α4)3(ß2)2) isoforms of human α4ß2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4ß2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4ß2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4ß2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using ß2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4ß2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)ß2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4ß2-nAChR isoforms, and demonstrate that HS versus LS α4ß2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4ß2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.


Assuntos
Agonistas Nicotínicos/metabolismo , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/classificação , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Feminino , Humanos , Agonistas Nicotínicos/química , Oócitos/química , Oócitos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Xenopus laevis
10.
Bioorg Med Chem Lett ; 24(2): 674-8, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24365158

RESUMO

The design and synthesis of a series of substituted heteroaromatic α4ß2α5 positive allosteric modulators is reported. The optimization and development of the heteroaromatic series was carried out from NS9283, and several potent analogues, such as 3-(5-(pyridin-3-yl)-2H-tetrazol-2-yl)benzonitrile (5k) and 3,3'-(2H-tetrazole-2,5-diyl)dipyridine (12 h) with good in vitro efficacy were discovered.


Assuntos
Agonistas Nicotínicos/síntese química , Antagonistas Nicotínicos/síntese química , Receptores Nicotínicos/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Células HEK293 , Humanos , Camundongos , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Oxidiazóis/síntese química , Oxidiazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Relação Estrutura-Atividade
11.
Genomics Proteomics Bioinformatics ; 11(4): 207-18, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23891776

RESUMO

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and ß2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between α4ß2-containing (α4ß2(∗)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α4ß2(∗) nAChRs in a genedose dependent pattern by immunopurifying ß2(∗) nAChRs from mice that differ in α4 and ß2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the ß2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of α4ß2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a ß2(∗) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4ß2 nAChRs and their downstream signaling mechanisms.


Assuntos
Proteoma/análise , Proteômica/métodos , Receptores Nicotínicos/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia Líquida , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nicotina/farmacologia , Ligação Proteica , Proteoma/metabolismo , Receptores Nicotínicos/genética , Espectrometria de Massas em Tandem
12.
Pharmacol Biochem Behav ; 103(3): 603-21, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23123803

RESUMO

Several mutations in α4 or ß2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the ß2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (ß2VL). Previous studies indicated that the ß2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of ß2VL mice. No differences in ß2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the ß2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Nicotina/farmacologia , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/genética , Temperatura Corporal/fisiologia , Sensibilização do Sistema Nervoso Central/genética , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Dopamina/metabolismo , Distonia/induzido quimicamente , Distonia/genética , Distonia/fisiopatologia , Técnicas de Introdução de Genes , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Atividade Motora/fisiologia , Mutação de Sentido Incorreto/genética , Nicotina/administração & dosagem , Terminações Pré-Sinápticas/efeitos dos fármacos , Ensaio Radioligante/métodos , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Radioisótopos de Rubídio , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Convulsões/fisiopatologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Ácido gama-Aminobutírico/metabolismo
13.
J Pharmacol Exp Ther ; 337(1): 187-200, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21228066

RESUMO

Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of α4ß2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion. Tissue sections were prepared, and binding of [(125)I]3-((2S)-azetidinylmethoxy)-5-iodo-pyridine (A85380) to ß2*-nAChR sites, [(125)I]monoclonal antibody (mAb) 299 to α4 nAChR subunits, and [(125)I]mAb 270 to ß2 nAChR subunits was determined by quantitative autoradiography. Chronic nicotine treatment dose-dependently increased binding of all three ligands. In regions that express α4ß2-nAChR almost exclusively, binding of all three ligands increased coordinately. However, in brain regions containing significant ß2*-nAChR without α4 subunits, relatively less increase in mAb 270 binding to ß2 subunits was observed. Signal intensity measured with the mAbs was lower than that with [(125)I]A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [(125)I]epibatidine binding sites with mAb 270 in select regions of nicotine-treated mice was nearly quantitative, although somewhat less so with mAb 299, confirming that the mAbs effectively recognize their targets. The patterns of change measured using immunoprecipitation were comparable with those determined autoradiographically. Thus, increases in α4ß2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein.


Assuntos
Encéfalo/efeitos dos fármacos , Nicotina/administração & dosagem , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima/fisiologia
14.
Ann Neurol ; 67(4): 441-51, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20437579

RESUMO

OBJECTIVE: Myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), are antibody (Ab)-mediated autoimmune diseases, in which autoantibodies bind to and cause loss of muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. To develop a specific immunotherapy of MG, we treated rats with ongoing EAMG by intraperitoneal injection of bacterially-expressed human muscle AChR constructs. METHODS: Rats with ongoing EAMG received intraperitoneal treatment with the constructs weekly for 5 weeks beginning after the acute phase. Autoantibody concentration, subclassification, and specificity were analyzed to address the underlying therapeutic mechanism. RESULTS: EAMG was specifically suppressed by diverting autoantibody production away from pathologically relevant specificities directed at epitopes on the extracellular surface of muscle AChRs toward pathologically irrelevant epitopes on the cytoplasmic domain. A mixture of subunit cytoplasmic domains was more effective than a mixture containing both extracellular and cytoplasmic domains or than only the extracellular domain of alpha1 subunits. INTERPRETATION: Therapy using only cytoplasmic domains, which lack pathologically relevant epitopes, avoids the potential liability of boosting the pathological response. Use of a mixture of bacterially-expressed human muscle AChR cytoplasmic domains for antigen-specific immunosuppression of myasthenia gravis has the potential to be specific, robust, and safe.


Assuntos
Imunoterapia/métodos , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/terapia , Receptores Colinérgicos/imunologia , Animais , Autoanticorpos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/uso terapêutico , Músculo Esquelético/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Subunidades Proteicas/imunologia , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/química , Fatores de Tempo
15.
J Neurochem ; 110(6): 1885-94, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19627445

RESUMO

Mechanisms that regulate early events in the biogenesis of the alpha7 nicotinic acetylcholine receptor (alpha7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the alpha7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric alpha7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH-SY5Y cells. Although the number of mature alpha7 AChRs is increased significantly in the presence of the chaperone protein resistant to inhibitors of cholineesterase-3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of alpha7 subunits are aggregated or improperly assembled. Transfection of alpha7 AChRs in SH-SY5Y cells, which endogenously express the alpha7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of alpha7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes.


Assuntos
Mutação/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Aconitina/análogos & derivados , Aconitina/metabolismo , Sequência de Aminoácidos , Animais , Bungarotoxinas/metabolismo , Linhagem Celular , Galinhas , Expressão Gênica/genética , Humanos , Imunoprecipitação/métodos , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucina/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Ligação Proteica/efeitos dos fármacos , Radioisótopos/metabolismo , Relação Estrutura-Atividade , Transfecção/métodos , Receptor Nicotínico de Acetilcolina alfa7
16.
J Biol Chem ; 284(35): 23251-9, 2009 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-19567877

RESUMO

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between alpha4beta2 AChRs and the presynaptic cell adhesion molecule, neurexin-1beta. In non-neuronal tsA 201 cells, recombinant neurexin-1beta and mature alpha4beta2 AChRs form complexes. alpha4beta2 AChRs and neurexin-1beta also coimmunoprecipitate from rat brain lysates. When exogenous alpha4beta2 AChRs and neurexin-1beta are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1beta. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1beta lacking the extracellular domain. Additionally, when alpha4beta2 AChRs, alpha7 AChRs, and neurexin-1beta are coexpressed in the same neuron, only the alpha4beta2 AChR colocalizes with neurexin-1beta at presynaptic terminals. Collectively, these data suggest that neurexin-1beta targets alpha4beta2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on alpha4beta2 AChRs, may contribute to the etiology of these neurological disorders.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Células Cultivadas , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Receptores Nicotínicos/genética
17.
Acta Pharmacol Sin ; 30(6): 795-804, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19498420

RESUMO

AIM: alpha2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of alpha2* nAChRs in these mouse brain regions. METHODS: alpha2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of alpha2 subunit-containing (alpha2*) nAChRs in these two regions. RESULTS: [(125)I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that alpha2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8+/-1.7 and 92+/-26 fmol mg(-1) protein, respectively). Pharmacological characterization using the beta2-selective drug A85380 to inhibit [(125)I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize alpha2* nAChRs. Protocols were established to immunoprecipitate beta2 and beta4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from beta2- and beta4-null mutant mice, and efficacy was good (>90% of beta2* and >80% of beta4* nAChRs were routinely recovered). CONCLUSION: Immunoprecipitation experiments indicated that interpeduncular nucleus alpha2* nAChRs predominantly contain beta2 subunits, while those in olfactory bulbs contain mainly beta4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both beta2 and beta4 subunits.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Receptores Nicotínicos/metabolismo , Animais , Autorradiografia/métodos , Azetidinas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Agonistas Nicotínicos/farmacologia , Bulbo Olfatório/metabolismo , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/genética
18.
Invest Ophthalmol Vis Sci ; 50(3): 1408-15, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18952912

RESUMO

PURPOSE: The purpose of this study was to detect and establish the cellular localizations of nicotinic acetylcholine receptor (nAChR) subunits in Rhesus monkey retina. METHODS: Retinas were dissected from the eyes of monkeys killed after unrelated experiments. RNA was extracted and analyzed by RT-PCR, using primers designed against human sequences of alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits. The RT-PCR products were separated by gel electrophoresis and sequenced. Frozen sections of postmortem fixed monkey eyes were immunolabeled with well-characterized and specific monoclonal antibodies against the alpha3, alpha4, alpha6, alpha7, beta2, or beta4 nAChR subunits and visualized with fluorescence labeling. RESULTS: Products of the predicted size for the alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits were detected by RT-PCR in Rhesus monkey retina. Homology between transcripts from monkey retina and human nucleotide sequences ranged from 93 to 99%. Immunohistochemical studies demonstrated that neurons in various cell layers of monkey retina expressed alpha3, alpha4, alpha7, or beta2 nAChR subunits and cells with the morphology of microglia were immunoreactive for the alpha6 or beta4 nAChR subunits. CONCLUSIONS: nAChR subunits are expressed in the monkey retina and localize to diverse retinal neurons as well as putative microglia. Besides mediating visual processing, retinal nAChRs may influence refractive development and ocular pathologies such as neovascularization.


Assuntos
Receptores Nicotínicos/metabolismo , Retina/metabolismo , Animais , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Macaca mulatta , Microscopia de Fluorescência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , Receptores Nicotínicos/genética , Neurônios Retinianos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Proc Natl Acad Sci U S A ; 104(51): 20570-5, 2007 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-18077321

RESUMO

Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The beta2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the beta2 subunit and components of immunoprecipitated beta2-nAChR complexes from mouse brain. Using the beta2-knockout mouse to exclude nonspecific binding to the beta2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the beta2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the beta2-nAChR. Interactions of the beta2 subunit with NIPs such as G protein alpha, G protein-regulated inducer of neurite outgrowth 1, and G protein-activated K(+) channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the beta2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.


Assuntos
Encéfalo/metabolismo , Proteômica , Receptores Nicotínicos/metabolismo , Animais , Imunoprecipitação , Camundongos , Camundongos Knockout , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteínas/análise , Proteínas/metabolismo , Receptores Nicotínicos/análise , Receptores Nicotínicos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
J Neurosci ; 27(31): 8202-18, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17670967

RESUMO

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change alpha4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional alpha4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases alpha4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated alpha4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional alpha4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.


Assuntos
Tolerância a Medicamentos/fisiologia , Potenciação de Longa Duração/fisiologia , Mesencéfalo/metabolismo , Nicotina/administração & dosagem , Via Perfurante/metabolismo , Receptores Nicotínicos/biossíntese , Animais , Relação Dose-Resposta a Droga , Potenciação de Longa Duração/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Via Perfurante/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
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