RESUMO
Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.
Assuntos
Músculos/metabolismo , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/metabolismo , Toxinas Biológicas/metabolismo , Viperidae/genética , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , Humanos , Músculos/citologia , Neurônios/citologia , Proteínas Recombinantes/genética , Toxinas Biológicas/genéticaRESUMO
Fluorescent derivatives are widely used to study the structure and functions of proteins. Quantum dots (QDs), fluorescent semiconductor nanocrystals, have a high quantum yield and are much more resistant to bleaching compared to organic dyes. Conjugates of α-neurotoxins with QDs were used for visualization of human α7 acetylcholine receptors heterologously expressed in GH4C1 pituitary adenoma cells. Specific staining of cells by the conjugated toxins was observed.
Assuntos
Neurotoxinas/química , Neurotoxinas/metabolismo , Pontos Quânticos/química , Venenos de Serpentes/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Linhagem Celular Tumoral , Humanos , Imagem MolecularRESUMO
A role of nicotinic acetylcholine receptors (nAChR) in the development of Parkinson's disease (PD) has been investigated using two mouse models corresponding to the presymptomatic stage and the early symptomatic stage of PD. Quantitative determination of nAChR in the striatum and substantia nigra (SN) was performed using the radioactive derivatives of epibatidine, ï¡-conotoxin MII, and ï¡-bungarotoxin as ligands. The number of ligand-binding sites changed differently depending on their location in the brain, the stage of the disease and the receptor subtype. Epibatidine binding decreased in the striatum to 66% and 70% at the presymptomatic and early symptomatic stages, respectively, whereas in SN a 160% increase was registered at the presymptomatic stage. The ï¡-conotoxin MII binding on striatal dopaminergic axonal terminals at the presymptomatic stage decreased by 20% and at the symptomatic stage it demonstrated a further decrease. The increase in ï¡-bungarotoxin binding at the presymptomatic stage and a decrease at the early symptomatic stage was observed in the striatum. In SN, the level of ï¡-bungarotoxin binding decreased at the presymptomatic stage and kept constant at the symptomatic stage. The significant decrease in the expression of Chrna4 and Chrna6 genes encoding ï¡4 and ï¡6 nAChR subunits was observed in SN at the early symptomatic stage, while a 13-fold increase in expression of the Chrna7 gene encoding the ï¡7 nAChR subunit was detected at the presymptomatic stage. The data obtained suggest possible involvement of nAChR in compensatory mechanisms at early PD stages.
Assuntos
Corpo Estriado/metabolismo , Doença de Parkinson Secundária/genética , Receptores Nicotínicos/genética , Substância Negra/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Animais , Doenças Assintomáticas , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bungarotoxinas/farmacologia , Conotoxinas/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Ligantes , Camundongos , Agonistas Nicotínicos/farmacologia , Especificidade de Órgãos , Doença de Parkinson Secundária/metabolismo , Doença de Parkinson Secundária/fisiopatologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de Sinais , Substância Negra/efeitos dos fármacos , Substância Negra/fisiopatologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
We studies the receptor-binding specificity of the synthetic peptide HAP (High Affinity Peptide) and its analogues, which are regarded as a model of the orthosteric site nicotinic acetylcholine receptors (nAChR). Using radioligand analysis, electrophysiology tests, and calcium imaging, we assessed the ability of HAP to interact with nAChR antagonists: long α-neurotoxins and α-conotoxins. A high affinity of HAP for α-bungarotoxin and the absence of its interaction with α-cobratoxin and α-conotoxins was found. The synthesized analogues of HAP in general retained the properties of the original peptide. Thus, HAP cannot be a model of a ligand-binding site.
Assuntos
Colinérgicos/farmacologia , Fragmentos de Peptídeos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Conotoxinas/metabolismo , Conotoxinas/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Modelos Moleculares , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Oócitos , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Ensaio Radioligante , Ratos , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Torpedo , Imagens com Corantes Sensíveis à Voltagem , Xenopus laevisRESUMO
With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 µM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.
Assuntos
Proteínas de Bactérias/metabolismo , Encéfalo/metabolismo , Proteínas Neurotóxicas de Elapídeos/metabolismo , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Aplysia , Proteínas de Bactérias/química , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Cianobactérias , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/química , Drosophila melanogaster , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Elapidae , Escherichia coli , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície , Receptor Nicotínico de Acetilcolina alfa7/químicaAssuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Doença de Parkinson/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Encéfalo/patologia , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Doença de Parkinson/patologiaAssuntos
Proteínas de Algas/metabolismo , Diatomáceas/metabolismo , Diatomáceas/ultraestrutura , Água Doce , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Algas/química , Sequência de Aminoácidos , Transporte Biológico , Immunoblotting , Microscopia Imunoeletrônica , Dados de Sequência MolecularRESUMO
We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.
