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1.
J Neurosci ; 41(3): 555-575, 2021 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-33239400

RESUMEN

Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-ß (Aß) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-ß (oAß42), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAß42 activates both homomeric α7- and heteromeric α7ß2-nAChR subtypes while preferentially enhancing α7ß2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAß42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the ß2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7ß2-nAChR in mediating the effects of oAß42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7ß2-nAChR in oAß42-induced cognitive decline.


Asunto(s)
Péptidos beta-Amiloides/genética , Prosencéfalo Basal/fisiopatología , Disfunción Cognitiva/genética , Disfunción Cognitiva/fisiopatología , Sistema Nervioso Parasimpático/fisiopatología , Fragmentos de Péptidos/genética , Transducción de Señal/genética , Receptor Nicotínico de Acetilcolina alfa 7/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Línea Celular , Fenómenos Electrofisiológicos , Femenino , Genotipo , Humanos , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Transgénicos , Neuronas/patología
2.
Acta Pharmacol Sin ; 43(4): 889-896, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34253876

RESUMEN

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.


Asunto(s)
Nicotina , Receptores Nicotínicos , Alcaloides de Berberina , Humanos , Cinética , Nicotina/farmacología , Receptores Nicotínicos/metabolismo
3.
Acta Pharmacol Sin ; 41(2): 163-172, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31399700

RESUMEN

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3ß4-nAChR expressed in native SH-SY5Y cells. α3ß4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 µM. Kinetic analysis showed that cocaine accelerated α3ß4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 µM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-ßS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 µM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3ß2ß3-nAChRs and α4ß2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3ß4-nAChRs expressed in SH-SY5Y cells.


Asunto(s)
Cocaína/farmacología , Neuronas/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Línea Celular Tumoral , Cocaína/administración & dosificación , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Neuroblastoma/metabolismo , Neuronas/metabolismo , Nicotina/farmacología , Técnicas de Placa-Clamp , Receptores Nicotínicos/metabolismo
4.
Stroke ; 49(10): 2445-2452, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30355112

RESUMEN

Background and Purpose- Tobacco cigarette smoking is considered to be a strong risk factor for intracranial aneurysmal rupture. Nicotine is a major biologically active constituent of tobacco products. Nicotine's interactions with vascular cell nicotinic acetylcholine receptors containing α7 subunits (α7*-nAChR) are thought to promote local inflammation and sustained angiogenesis. In this study, using a mouse intracranial aneurysm model, we assessed potential contributions of nicotine exposure and activation of α7*-nAChR to the development of aneurysmal rupture. Methods- Intracranial aneurysms were induced by a combination of deoxycorticosterone-salt induced hypertension and a single-dose elastase injection into cerebrospinal fluid in mice. Results- Exposure to nicotine or an α7*-nAChR-selective agonist significantly increased aneurysm rupture rate. Coexposure to an α7*-nAChR antagonist abolished nicotine's deleterious effect. In addition, nicotine's promotion of aneurysm rupture was absent in smooth muscle cell-specific α7*-nAChR subunit knockout mice but not in mice lacking α7*-nAChR on endothelial cells or macrophages. Nicotine treatment increased the mRNA levels of vascular endothelial growth factor, platelet-derived growth factor-B, and inflammatory cytokines. α7*-nAChR antagonist reversed nicotine-induced upregulation of these growth factors and cytokines. Conclusions- Our findings indicate that nicotine exposure promotes aneurysmal rupture through actions on vascular smooth muscle cell α7*-nAChR.


Asunto(s)
Aneurisma Roto/tratamiento farmacológico , Aneurisma Intracraneal/tratamiento farmacológico , Nicotina/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/efectos de los fármacos , Animales , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ratones Transgénicos , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Regulación hacia Arriba/efectos de los fármacos
5.
FASEB J ; 31(4): 1398-1420, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28100642

RESUMEN

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.


Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Receptores Nicotínicos/metabolismo , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales , Animales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Proteínas Ligadas a GPI/genética , Humanos , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Xenopus
6.
Acta Pharmacol Sin ; 39(10): 1571-1581, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29795357

RESUMEN

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4ß2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with ß2 and ß3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4ß2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-ß-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4ß2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.


Asunto(s)
Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Cinética , Técnicas de Placa-Clamp/métodos , Receptores Nicotínicos/química
7.
J Biol Chem ; 291(5): 2444-59, 2016 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-26644472

RESUMEN

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.


Asunto(s)
Regulación de la Expresión Génica , Receptores Nicotínicos/química , Acetilcolina/química , Sitio Alostérico , Animales , Azetidinas/química , Sitios de Unión/genética , ADN Complementario/metabolismo , Electrofisiología , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Nicotina/química , Agonistas Nicotínicos/química , Oocitos/metabolismo , Técnicas de Placa-Clamp , Unión Proteica , Isoformas de Proteínas , ARN/metabolismo , Xenopus laevis
8.
Biochem Biophys Res Commun ; 473(4): 795-800, 2016 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-27049309

RESUMEN

Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4ß2 nAChR, α1ß2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1ß2γ2 GABAA receptor.


Asunto(s)
Disacáridos/farmacología , Activación del Canal Iónico/fisiología , Ivermectina/análogos & derivados , Receptores de GABA/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Insecticidas/farmacología , Ivermectina/farmacología , Agonistas Nicotínicos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Receptores de GABA/metabolismo , Receptores de GABA-A , Receptores Nicotínicos/metabolismo , Xenopus laevis
9.
J Biol Chem ; 289(41): 28338-51, 2014 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-25028511

RESUMEN

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mß4hß3- (∼ 5-8-fold) or WT mα6mß4mß3-nAChRs (∼ 2-fold) yielded higher function than mα6mß4-nAChRs. Function was not detected when mα6 and mß2 subunits were expressed together or in the additional presence of hß3 or mß3 subunits. However, function emerged upon expression of mα6mß2mß3(V9'S)-nAChRs containing ß3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when hß3(V9'S) subunits were substituted for mß3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human ß3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hß3 subunits as templates for site-directed mutagenesis studies, substitution with mß3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "ß2-ß3" (Gln(94) and Glu(101)) increased function of mα6mß2*- (∼ 2-3-fold) or mα6mß4* (∼ 2-4-fold)-nAChRs. EC50 values for nicotine acting at mα6mß4*-nAChR were unaffected by ß3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops ß2-ß3 and E) interfaces of ß3 subunits are some of the molecular impediments for functional expression of mα6mß2ß3- or mα6mß4ß3-nAChRs.


Asunto(s)
Aminoácidos/química , Subunidades de Proteína/química , Receptores Nicotínicos/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Atropina/metabolismo , Atropina/farmacología , Femenino , Expresión Génica , Humanos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Nicotina/metabolismo , Nicotina/farmacología , Oocistos/citología , Oocistos/efectos de los fármacos , Oocistos/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevis
10.
J Neurosci ; 33(17): 7253-63, 2013 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-23616534

RESUMEN

There is a significantly elevated incidence of epilepsy in Alzheimer's disease (AD). Moreover, there is neural hyperexcitation/synchronization in transgenic mice expressing abnormal levels or forms of amyloid precursor protein and its presumed, etiopathogenic product, amyloid-ß1-42 (Aß). However, the underlying mechanisms of how Aß causes neuronal hyperexcitation remain unclear. Here, we report that exposure to pathologically relevant levels of Aß induces Aß form-dependent, concentration-dependent, and time-dependent neuronal hyperexcitation in primary cultures of mouse hippocampal neurons. Similarly, Aß exposure increases levels of nicotinic acetylcholine receptor (nAChR) α7 subunit protein on the cell surface and α7-nAChR function, but not α7 subunit mRNA, suggesting post-translational upregulation of functional α7-nAChRs. These effects are prevented upon coexposure to brefeldin A, an inhibitor of endoplasmic reticulum-to-Golgi protein transport, consistent with an effect on trafficking of α7 subunits and assembled α7-nAChRs to the cell surface. Aß exposure-induced α7-nAChR functional upregulation occurs before there is expression of neuronal hyperexcitation. Pharmacological inhibition using an α7-nAChR antagonist or genetic deletion of nAChR α7 subunits prevents induction and expression of neuronal hyperexcitation. Collectively, these results, confirmed in studies using slice cultures, indicate that functional activity and perhaps functional upregulation of α7-nAChRs are necessary for production of Aß-induced neuronal hyperexcitation and possibly AD pathogenesis. This novel mechanism involving α7-nAChRs in mediation of Aß effects provides potentially new therapeutic targets for treatment of AD.


Asunto(s)
Péptidos beta-Amiloides/fisiología , Hipocampo/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/fisiología , Receptores Nicotínicos/biosíntesis , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Hipocampo/citología , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7
11.
Mol Pharmacol ; 86(3): 306-17, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25002271

RESUMEN

We examined α7ß2-nicotinic acetylcholine receptor (α7ß2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7ß2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7ß2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using ß2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(ß2)1-, and (α7)3(ß2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(ß2)1-, and (α7)3(ß2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7ß2- versus α7-nAChRs. This study represents the first direct confirmation of α7ß2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7ß2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7ß2-nAChR subunit isoforms with different α7/ß2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7ß2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.


Asunto(s)
Prosencéfalo/metabolismo , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bungarotoxinas/farmacología , Cerebelo/metabolismo , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Oocitos/metabolismo , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Piridinas/farmacología , Ensayo de Unión Radioligante , Receptores Nicotínicos/genética , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/genética
12.
J Pharmacol Exp Ther ; 348(1): 46-58, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24190916

RESUMEN

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.


Asunto(s)
Agonistas Nicotínicos/metabolismo , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/clasificación , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Sitios de Unión/fisiología , Línea Celular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Agonistas Nicotínicos/química , Oocitos/química , Oocitos/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Receptores Nicotínicos/genética , Xenopus laevis
13.
Br J Pharmacol ; 181(17): 3172-3191, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38720171

RESUMEN

BACKGROUND AND PURPOSE: Oligomeric amyloid ß 1-42 (oAß1-42) exhibits agonist-like action at human α7- and α7ß2-containing nicotinic receptors. The N-terminal amyloid ß1-15 fragment (N-Aß fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aß fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aßcore), protect against oAß1-42-associated synapto- and neurotoxicity. Here, we investigated how oAß1-42, the N-Aß fragment, and the N-Aßcore regulate the single-channel properties of α7- and α7ß2-nicotinic receptors. EXPERIMENTAL APPROACH: Single-channel recordings measured the impact of acetylcholine, oAß1-42, the N-Aß fragment, and the N-Aßcore on the unitary properties of human α7- and α7ß2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aß fragment and orthosteric α7+/α7- and α7+/ß2- nicotinic receptor binding interfaces. KEY RESULTS: The N-Aß fragment and N-Aßcore induced α7- and α7ß2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAß1-42 preferentially enhanced α7ß2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aßcore neutralized these effects. Further, administration of the N-Aß fragment alone, or in combination with acetylcholine or oAß1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAß1-42). CONCLUSIONS AND IMPLICATIONS: Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7ß2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7ß2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid ß-derived fragments against the toxic effects of oAß1-42.


Asunto(s)
Péptidos beta-Amiloides , Fragmentos de Péptidos , Receptor Nicotínico de Acetilcolina alfa 7 , Humanos , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/toxicidad , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Fragmentos de Péptidos/farmacología , Fármacos Neuroprotectores/farmacología , Simulación de Dinámica Molecular , Acetilcolina/farmacología , Receptores Nicotínicos/metabolismo
14.
J Neurosci ; 32(36): 12366-75, 2012 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-22956827

RESUMEN

Systemic administration of nicotine increases dopaminergic (DA) neuron firing in the ventral tegmental area (VTA), which is thought to underlie nicotine reward. Here, we report that the medial prefrontal cortex (mPFC) plays a critical role in nicotine-induced excitation of VTA DA neurons. In chloral hydrate-anesthetized rats, extracellular single-unit recordings showed that VTA DA neurons exhibited two types of firing responses to systemic nicotine. After nicotine injection, the neurons with type-I response showed a biphasic early inhibition and later excitation, whereas the neurons with type-II response showed a monophasic excitation. The neurons with type-I, but not type-II, response exhibited pronounced slow oscillations (SOs) in firing. Pharmacological or structural mPFC inactivation abolished SOs and prevented systemic nicotine-induced excitation in the neurons with type-I, but not type-II, response, suggesting that these VTA DA neurons are functionally coupled to the mPFC and nicotine increases firing rate in these neurons in part through the mPFC. Systemic nicotine also increased the firing rate and SOs in mPFC pyramidal neurons. mPFC infusion of a non-α7 nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the excitatory effect of systemic nicotine on the VTA DA neurons with type-I response, but mPFC infusion of nicotine failed to excite these neurons. These results suggest that nAChR activation in the mPFC is necessary, but not sufficient, for systemic nicotine-induced excitation of VTA neurons. Finally, systemic injection of bicuculline prevented nicotine-induced firing alterations in the neurons with type-I response. We propose that the mPFC plays a critical role in systemic nicotine-induced excitation of VTA DA neurons.


Asunto(s)
Anestesia , Neuronas Dopaminérgicas/fisiología , Nicotina/farmacología , Corteza Prefrontal/fisiología , Área Tegmental Ventral/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Anestesia/métodos , Animales , Neuronas Dopaminérgicas/efectos de los fármacos , Masculino , Corteza Prefrontal/efectos de los fármacos , Ratas , Ratas Wistar , Área Tegmental Ventral/efectos de los fármacos
15.
J Biol Chem ; 287(17): 14259-69, 2012 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-22315221

RESUMEN

We previously have shown that ß3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6ß4ß3-nAChR) or potentiate (e.g. for hybrid mα6hß4hß3- or mα6mß4hß3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hß3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of ß3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9' or 13' positions (L9'S or V13'S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of ß3 subunit incorporation. However, coexpression with ß3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6((L9'S or V13'S))ß4*- or α6(N143D+M145V)(L9'S)ß2*-nAChR. This contrasts with the lack of consistent function when α6((L9'S or V13'S)) and ß2 subunits are expressed alone or in the presence of wild-type ß3 subunits. These results provide evidence that gain-of-function hα6hß2*-nAChR (i.e. hα6(N143D+M145V)(L9'S)hß2hß3 nAChR) could be produced in vitro. These studies also indicate that nAChR ß3 subunits can be assembly partners in functional α6*-nAChR and that 9' or 13' mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that ß3 subunit coexpression promotes function of α6*-nAChR.


Asunto(s)
Receptores Nicotínicos/fisiología , Animales , Clonación Molecular , Electrofisiología/métodos , Femenino , Humanos , Iones/química , Ligandos , Ratones , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Xenopus laevis
16.
J Biol Chem ; 287(30): 25151-62, 2012 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-22665477

RESUMEN

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3ß4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, ß4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3ß4* nAChRs. α5 subunit incorporation reduces α3ß4* nAChR function after coinjection with unlinked α3 and ß4 subunits but increases that of α3ß4α5 versus α3ß4-only concatemers. α5 subunit incorporation into α3ß4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and ß4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3ß4-only subtypes are dissimilar both to each other and to those of α3ß4α5 nAChR. The α5 variant-induced change in α3ß4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.


Asunto(s)
Sustitución de Aminoácidos , Complejos Multiproteicos/metabolismo , Mutación Missense , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Transducción de Señal/fisiología , Animales , Estudio de Asociación del Genoma Completo , Humanos , Complejos Multiproteicos/genética , Oocitos , Polimorfismo Genético , Subunidades de Proteína/genética , Receptores Nicotínicos/genética , Xenopus laevis
17.
Immunol Cell Biol ; 91(3): 195-200, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23399696

RESUMEN

Nicotine is a potent inhibitor of the immune response and is protective against experimental autoimmune encephalomyelitis (EAE). Initial studies suggested that the cholinergic system modulates inflammation via the α7-nicotinic acetylcholine receptor (nAChR) subtype. We recently have shown that effector T cells and myeloid cells constitutively express mRNAs encoding nAChR α9 and ß2 subunits and found evidence for immune system roles for non-α7-nAChRs. In the present study, we assessed the effects of nAChR α9 or ß2 subunit gene deletion on EAE onset and severity, with or without nicotine treatment. We report again that disease onset is delayed and severity is attenuated in nicotine-treated, wild-type mice, an effect that also is observed in α9 subunit knock-out (KO) mice irrespective of nicotine treatment. On the other hand, ß2 KO mice fail to recover from peak measures of disease severity regardless of nicotine treatment, despite retaining sensitivity to nicotine's attenuation of disease severity. Prior to disease onset, we found significantly less reactive oxygen species production in the central nervous system (CNS) of ß2 KO mice, elevated proportions of CNS myeloid cells but decreased ratios of CNS macrophages/microglia in α9 or ß2 KO mice, and some changes in iNOS, TNF-α and IL-1ß mRNA levels in α9 KO and/or ß2 KO mice. Our data thus suggest that ß2*- and α9*-nAChRs, in addition to α7-nAChRs, have different roles in endogenous and nicotine-dependent modulation of immune functions and could be exploited as therapeutic targets to modulate inflammation and autoimmunity.


Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Macrófagos/inmunología , Microglía/inmunología , Receptores Nicotínicos/inmunología , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Eliminación de Gen , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Macrófagos/patología , Ratones , Ratones Noqueados , Microglía/patología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Especies Reactivas de Oxígeno/inmunología , Receptores Nicotínicos/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología
18.
J Neurosci ; 31(7): 2537-48, 2011 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-21325521

RESUMEN

Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for α6ß2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA(A) receptors. These increases are abolished by α6*-nAChR-selective α-Ctx MII or α-Ctx PIA (1 nm) but not by α7 (10 nm methyllycaconitine) or α4* (1 µm dihydro-ß-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR ß2 knock-out mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca(2+) in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that α6ß2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6ß2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.


Asunto(s)
Dopamina/metabolismo , Neuronas/citología , Terminales Presinápticos/fisiología , Receptores Nicotínicos/metabolismo , Área Tegmental Ventral/citología , Ácido gamma-Aminobutírico/metabolismo , 4-Aminopiridina/farmacología , Acetilcolina/farmacología , Aconitina/análogos & derivados , Aconitina/farmacología , Animales , Animales Recién Nacidos , Bungarotoxinas/farmacología , Calcio/metabolismo , Conotoxinas/metabolismo , Dihidro-beta-Eritroidina/farmacología , Dopamina/farmacología , Interacciones Farmacológicas , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Femenino , GABAérgicos/farmacología , Glutamato Descarboxilasa/metabolismo , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Nicotina/farmacología , Antagonistas Nicotínicos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Nicotínicos/deficiencia , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo
19.
Mol Pharmacol ; 81(2): 175-88, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22039094

RESUMEN

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and ß2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and ß2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and ß2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and ß2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7ß2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7ß2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for α7 nAChRs. It is noteworthy that α7ß2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-ß-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-ß2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7ß2 nAChR current amplitudes and challenges in identifying the function of native α7ß2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of ß2 subunits within the α7ß2 nAChRs.


Asunto(s)
Fenómenos Electrofisiológicos , Multimerización de Proteína , Receptores Nicotínicos/fisiología , Proteínas de Xenopus/fisiología , Animales , Línea Celular , Electrofisiología , Oocitos , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7
20.
J Biol Chem ; 286(44): 37905-37918, 2011 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-21873428

RESUMEN

To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/ß3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human ß3 subunit) almost completely abolishes the very small amount of function seen for α6ß4*-nAChR and does not induce function of α6ß2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6ß4*-nAChR or induce function of α6ß2*-nAChR. However, coexpression with mutant chimeric α5/ß3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6ß4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/ß3(V9)'(S) subunits to have a gain-of-function effect on α6ß2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either ß2 or ß4 subunits have some function that is modulated in the presence of α5 or α5/ß3 subunits. Coexpression with α5/ß3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.


Asunto(s)
Mutación , Proteínas del Tejido Nervioso/genética , Receptores Nicotínicos/genética , Animales , Citoplasma/metabolismo , ADN Complementario/metabolismo , Electrofisiología/métodos , Femenino , Humanos , Ligandos , Proteínas del Tejido Nervioso/metabolismo , Nicotina/metabolismo , Oocitos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión/química , Xenopus laevis
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