Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , GMP Cíclico/fisiologia , Canais Iônicos/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacocinética , Animais , Bovinos , Linhagem Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacocinética , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Proteínas do Olho/fisiologia , Fura-2 , Humanos , Canais Iônicos/química , Cinética , Modelos Biológicos , Técnicas de Patch-Clamp , Proteínas Recombinantes/metabolismo , Segmento Externo da Célula Bastonete/fisiologia , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , TransfecçãoRESUMO
Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.
Assuntos
Cálcio/metabolismo , Canais Iônicos/metabolismo , Animais , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Bovinos , Linhagem Celular , Permeabilidade da Membrana Celular , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Drosophila melanogaster , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/genética , Cinética , Potenciais da Membrana , Nucleotídeos Cíclicos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Conformação Proteica , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoAssuntos
AMP Cíclico/análogos & derivados , GMP Cíclico/análogos & derivados , Fotoquímica , Animais , Bovinos , Linhagem Celular , AMP Cíclico/síntese química , AMP Cíclico/química , AMP Cíclico/efeitos da radiação , GMP Cíclico/síntese química , GMP Cíclico/química , GMP Cíclico/efeitos da radiação , Desenho de Fármacos , Humanos , Hidrólise , Canais Iônicos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Fotólise , Células Fotorreceptoras Retinianas Cones/metabolismo , SolubilidadeRESUMO
In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels.
Assuntos
Células Fotorreceptoras/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/metabolismo , Potássio/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Bovinos , Condutividade Elétrica , Canais de Potássio Éter-A-Go-Go , Isomerismo , Cinética , Magnésio/fisiologia , Dados de Sequência Molecular , Nucleotídeos Cíclicos/fisiologia , Permeabilidade , Canais de Potássio/fisiologia , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The application of the 1-(2-nitrophenyl)ethyl (NPE) moiety as a photolabile ligand for the release of hydrolysis-resistant 8-Br-cAMP and 8-Br-cGMP was examined. NPE-caged 8-Br-cAMP and 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light. The synthesis procedure resulted in diastereoisomeric mixtures, which were chromatographically separated into the axial and equatorial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP. The hydrolytic stability, solubility and photochemical properties of these derivatives were compared to the previously reported 4.5-dimethoxy-2-nitrobenzyl (DMNB) compounds. We found that the axial isomers of NPE-caged 8-Br-cAMP and 8-Br-cGMP had a considerably better solvolytic stability than the respective equatorial isomers as well as the DMNB-caged derivatives. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/química , AMP Cíclico/análogos & derivados , GMP Cíclico/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Animais , Bovinos , Linhagem Celular , AMP Cíclico/química , GMP Cíclico/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Ésteres , Hidrólise , Canais Iônicos/metabolismo , Estrutura Molecular , Fotoquímica , FotóliseRESUMO
Photolabile compounds which rapidly release cAMP or cGMP after photolysis are widely used for in situ studies of signaling pathways inside cells. We synthesized two novel caged compounds, 4,5-dimethoxy-2-nitrobenzyl 8-Br-cAMP (caged 8-Br-cAMP) and 4,5-dimethoxy-2-nitrobenzyl 8-Br-cGMP caged 8-BR-cGMP), which respectively release the hydrolysis-resistant analogues 8-Br-cAMP and 8-Br-cGMP. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons. The synthesis procedure resulted in diastereomeric mixtures which were chromatographically separated into the axial and equatorial isomers of caged 8-BR-cAMP and of caged 8-BR-cGMP. The axial isomers which have a higher solubility and better solvolytic stability than the equatorial forms were used for experiments with CNG channels. Flashes of UV light produced steps in the concentration of 8-Br-cGMP which activated currents through CNG channels. Concentration steps inside the cell could be calibrated precisely using the relation between the ligand concentration and the normalized current. Similar results were obtained with caged 8-Br-cAMP. Control experiments with caged cGMP showed that flash-induced currents decayed within a few minutes because photoreleased cGMP was degraded by endogenous phosphodiesterase activity. The rise time of the 8-Br-cGMP-activated whole-cell current was consistent with a bimolecular reaction between channel and ligand.