RESUMO
The cholinergic α7 nicotinic receptor gene, CHRNA7, encodes a subunit that forms the homopentameric α7 receptor, involved in learning and memory. In humans, exons 5-10 in CHRNA7 are duplicated and fused to the FAM7A genetic element, giving rise to the hybrid gene CHRFAM7A Its product, dupα7, is a truncated subunit lacking part of the N-terminal extracellular ligand-binding domain and is associated with neurological disorders, including schizophrenia, and immunomodulation. We combined dupα7 expression on mammalian cells with patch clamp recordings to understand its functional role. Transfected cells expressed dupα7 protein, but they exhibited neither surface binding of the α7 antagonist α-bungarotoxin nor responses to acetylcholine (ACh) or to an allosteric agonist that binds to the conserved transmembrane region. To determine whether dupα7 assembles with α7, we generated receptors comprising α7 and dupα7 subunits, one of which was tagged with conductance substitutions that report subunit stoichiometry and monitored ACh-elicited channel openings in the presence of a positive allosteric α7 modulator. We found that α7 and dupα7 subunits co-assemble into functional heteromeric receptors, which require at least two α7 subunits for channel opening, and that dupα7's presence in the pentameric arrangement does not affect the duration of the potentiated events compared with that of α7. Using an α7 subunit mutant, we found that activation of (α7)2(dupα7)3 receptors occurs through ACh binding at the α7/α7 interfacial binding site. Our study contributes to the understanding of the modulation of α7 function by the human specific, duplicated subunit, associated with human disorders.
Assuntos
Acetilcolina/metabolismo , Bungarotoxinas/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, ß2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4ß2) and [¹²5I]-α-bungarotoxin ([¹²5I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²5I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²5I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, ß2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients.
Assuntos
Distrofina/deficiência , Hipocampo/metabolismo , Receptores Nicotínicos/metabolismo , Fatores Etários , Alcaloides/farmacocinética , Análise de Variância , Animais , Azocinas/farmacocinética , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacocinética , Relação Dose-Resposta a Droga , Distrofina/genética , Hipocampo/efeitos dos fármacos , Isótopos/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Antagonistas Nicotínicos/farmacocinética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Quinolizinas/farmacocinética , RNA Mensageiro/metabolismo , Receptores Nicotínicos/genéticaRESUMO
The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor. After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a Kd1 value of 29.6+/-5.72 nM and a Kd2 value of 315.9+/-26.6 nM, which is similar to the Kd value reported for the alpha4 nicotinic receptor. The Hill coefficients were determined to be an n1 of 1.2+/-0.3 and an n2 of 4.2+/-1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals.
Assuntos
Cálcio/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Trypanosoma/metabolismo , Animais , Bungarotoxinas/metabolismo , Relação Dose-Resposta a Droga , Masculino , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Distribuição Normal , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/efeitos dos fármacos , Trypanosoma/efeitos dos fármacosRESUMO
Bucain, a potent neurotoxin isolated from the venom of the Malayan krait (Bungarus candidus), induces paralysis and death. Its crystal structure has been determined at 2.10 A resolution and based on the molecular topology and hydrophobicity profile is structurally classified as a three-fingered alpha-neurotoxin possessing a positively charged AChR-binding site.
Assuntos
Bungarotoxinas/química , Bungarus/metabolismo , Neurotoxinas/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bungarotoxinas/isolamento & purificação , Bungarotoxinas/metabolismo , Cristalização , Cristalografia por Raios X , Venenos Elapídicos/química , Venenos Elapídicos/isolamento & purificação , Venenos Elapídicos/metabolismo , Dados de Sequência Molecular , Neurotoxinas/isolamento & purificação , Neurotoxinas/metabolismo , Estrutura Secundária de Proteína , Receptores Colinérgicos/metabolismo , Alinhamento de Sequência , Toxinas Biológicas/isolamento & purificação , Toxinas Biológicas/metabolismoRESUMO
Polybia-MPII (INWLKLGKMVIDAL-NH2), a mastoparan isolated from the crude venom of the swarming wasp Polybia paulista, was injected into the left hind limb of Swiss white mice. Between 3 h and 21 days later the mice were killed and the soleus muscles from both hind limbs were removed. Sections of the muscles were made for transmission electron microscopy and immunocytochemistry. Transmission electron microscopy showed that both the volume fraction occupied by synaptic vesicles and synaptic vesicle density was greatly reduced after exposure to Polybia-MPII, although there was no significant structural damage to the plasma membrane of the terminal boutons and mitochondria were indistinguishable from those in normal, control boutons. Immunocytochemistry revealed that in control muscles 99% of motor end plates identified by the positive labelling of acetylcholine receptors by TRITC-alpha-bungarotoxin co-labelled with anti-synaptophysin antibody, but this figure fell by 30% in muscles exposed to the toxin. These changes were transient. They were maximal at 6 h and fully reversed by 3 days. At no time was axonal labelling with anti-neurofilament antibodies affected by exposure to Polybia-MPII. We conclude that mastoparan Polybia-MPII is a minor neurotoxin and suggest that its neurotoxic activity is unlikely to be of clinical significance.
Assuntos
Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Neurotoxinas/toxicidade , Peptídeos/toxicidade , Vesículas Sinápticas/efeitos dos fármacos , Venenos de Vespas/toxicidade , Animais , Bungarotoxinas/metabolismo , Colinesterases/metabolismo , Injeções Intramusculares , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Músculo Esquelético/enzimologia , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/ultraestrutura , Receptores Nicotínicos/metabolismo , Rodaminas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismo , Vespas/químicaRESUMO
Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.
Assuntos
Sítios de Ligação , Cisteína/metabolismo , Agonistas Nicotínicos/metabolismo , Receptores Nicotínicos/química , Acetilcolina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Aminoácidos/genética , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Fenômenos Biofísicos/efeitos dos fármacos , Fenômenos Biofísicos/genética , Bungarotoxinas/metabolismo , Linhagem Celular Transformada , Cisteína/genética , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Expressão Gênica/fisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Técnicas de Patch-Clamp , Conformação Proteica , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores 5-HT3 de Serotonina/genética , Receptores 5-HT3 de Serotonina/metabolismo , Relação Estrutura-Atividade , Transfecção/métodos , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The anticonvulsive drug Lamotrigine (LTG) is found to activate adult muscle nicotinic acetylcholine receptors (AChR). Single-channel patch-clamp recordings showed that LTG (0.05-400 microM) applied alone is able to open AChR channels. [125I]alpha-bungarotoxin-binding studies further indicate that LTG does not bind to the canonical ACh-binding sites. Fluorescence experiments using the probe crystal violet demonstrate that LTG induces the transition from the resting state to the desensitized state of the AChR in the presence of excess alpha-bungarotoxin, that is, when the agonist site is blocked. Allosterically-potentiating ligands or the open-channel blocker QX-314 exhibited a behavior different from that of LTG. We conclude that LTG activates the AChR through a site that is different from those of full agonists/competitive antagonists and allosterically-potentiating ligands, respectively.
Assuntos
Anticonvulsivantes/metabolismo , Receptores Nicotínicos , Triazinas/metabolismo , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Lamotrigina , Antagonistas Nicotínicos/metabolismo , Técnicas de Patch-Clamp , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismoRESUMO
Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240-280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced ( approximately 50%) the number of receptor domains by accelerating the rate of endocytosis (t(1/2) decreased from 1.5-0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of Förster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/deficiência , Endocitose , Tamanho da Partícula , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Animais , Anticorpos/farmacologia , Bungarotoxinas/metabolismo , Células CHO , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Cricetinae , Cricetulus , Endocitose/efeitos dos fármacos , Canais Iônicos/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Fatores de Tempo , beta-Ciclodextrinas/farmacologiaRESUMO
The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 microM) was essentially without effect at DIV4 and DIV5, while dihydro-ss-erythroidine (10-100 microM) blocked the response to acetylcholine (3.0 nM-2.0 microM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.
Assuntos
Melatonina/farmacologia , Receptores Nicotínicos/biossíntese , Retina/metabolismo , Animais , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacologia , Células Cultivadas , Embrião de Galinha , Imuno-Histoquímica , Microquímica , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Retina/citologia , Retina/efeitos dos fármacos , Fatores de Tempo , Triptaminas/farmacologiaRESUMO
The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.