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1.
Proc Natl Acad Sci U S A ; 121(9): e2315132121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38377199

RESUMO

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Ligantes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , AMP Cíclico/metabolismo , Fenômenos Biofísicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo
2.
Biophys J ; 116(12): 2411-2422, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31130235

RESUMO

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.


Assuntos
AMP Cíclico/química , GMP Cíclico/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Corantes Fluorescentes/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Imagem Individual de Molécula
3.
PLoS Comput Biol ; 14(3): e1006045, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29565972

RESUMO

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control electrical rhythmicity in specialized brain and heart cells. We quantitatively analysed voltage-dependent activation of homotetrameric HCN2 channels and its modulation by the second messenger cAMP using global fits of hidden Markovian models to complex experimental data. We show that voltage-dependent activation is essentially governed by two separable voltage-dependent steps followed by voltage-independent opening of the pore. According to this model analysis, the binding of cAMP to the channels exerts multiple effects on the voltage-dependent gating: It stabilizes the open pore, reduces the total gating charge from ~8 to ~5, makes an additional closed state outside the activation pathway accessible and strongly accelerates the ON-gating but not the OFF-gating. Furthermore, the open channel has a much slower computed OFF-gating current than the closed channel, in both the absence and presence of cAMP. Together, these results provide detailed new insight into the voltage- and cAMP-induced activation gating of HCN channels.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Ativação do Canal Iônico/fisiologia , Animais , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Cinética , Cadeias de Markov , Modelos Neurológicos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Xenopus laevis/fisiologia
4.
Bioorg Med Chem ; 27(8): 1704-1713, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30879860

RESUMO

Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3',5'-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.


Assuntos
AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Adenina/análogos & derivados , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Animais , AMP Cíclico/síntese química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/agonistas , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Halogenação , Humanos , Camundongos
5.
Biophys J ; 109(11): 2268-76, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26636938

RESUMO

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3',5'-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels not preactivated by voltage. We identified a conformational flip of the channel as an intermediate state following the ligand binding and quantified it kinetically. Globally fitting the time courses of ligand binding and unbinding revealed modest cooperativity among the subunits in the conformational flip. The intensity of this cooperativity, however, was only moderate compared to channels preactivated by hyperpolarizing voltage. These data provide kinetic information about conformational changes proceeding in nonactivated HCN2 channels when cAMP binds. Moreover, our approach bears potential for analyzing the function of any other membrane receptor if a potent fluorescent ligand is available.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Nucleotídeos Cíclicos/farmacologia , Subunidades Proteicas/química , Animais , AMP Cíclico/metabolismo , Feminino , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Conformação Proteica , Subunidades Proteicas/metabolismo , Xenopus laevis
6.
Nat Chem Biol ; 8(2): 162-9, 2011 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-22179066

RESUMO

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.


Assuntos
Canais Iônicos/metabolismo , Animais , AMP Cíclico/farmacologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/química , Ligantes , Miócitos Cardíacos , Neurônios , Técnicas de Patch-Clamp , Canais de Potássio , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Subunidades Proteicas
7.
Biophys J ; 103(9): 1860-9, 2012 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-23199914

RESUMO

In a multimeric receptor protein, the binding of a ligand can modulate the binding of a succeeding ligand. This phenomenon, called cooperativity, is caused by the interaction of the receptor subunits. By using a complex Markovian model and a set of parameters determined previously, we analyzed how the successive binding of four ligands leads to a complex cooperative interaction of the subunits in homotetrameric HCN2 pacemaker channels. The individual steps in the model were characterized by Gibbs free energies for the equilibria and activation energies, specifying the affinity of the binding sites and the transition rates, respectively. Moreover, cooperative free energies were calculated for each binding step in both the closed and the open channel. We show that the cooperativity sequence positive-negative-positive determined for the binding affinity is generated by the combined effect of very different cooperativity sequences determined for the binding and unbinding rates, which are negative-negative-positive and no-negative-no, respectively. It is concluded that in the ligand-induced activation of HCN2 channels, the sequence of cooperativity based on the binding affinity is caused by two even qualitatively different sequences of cooperativity that are based on the rates of ligand binding and unbinding.


Assuntos
Canais Iônicos/metabolismo , Multimerização Proteica , Subunidades Proteicas/metabolismo , Animais , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Canais Iônicos/química , Cinética , Ligantes , Cadeias de Markov , Canais de Potássio , Ligação Proteica , Subunidades Proteicas/química
8.
J Cancer Res Clin Oncol ; 137(2): 261-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20396915

RESUMO

PURPOSE: As HER2 status is a strong predictor of the response to trastuzumab, clinical guidelines recommend that all breast tumours are first evaluated for HER2 protein expression by immunohistochemistry (IHC) followed by confirmatory testing for HER2 gene amplification using fluorescence in situ hybridisation (FISH) for 2+ cases. Alternatively, chromogenic in situ hybridisation (CISH) offers a simpler, less expensive approach to detect HER2 amplification. METHODS: In this prospective, multi-centre study, based on the largest dataset for HER2 testing in Germany to date, we evaluated the concordance between FISH and CISH in 399 samples from adjuvant and metastatic breast cancer patients. Tumour specimens from routine diagnostic practice were analysed by IHC, FISH and CISH in four reference centres. RESULTS: FISH and CISH results were strongly concordant (κ = 0.83), with 95% of cases showing agreement. Despite variable IHC scoring across testing centres, complete consensus among the three methods was observed for 246 cases, representing 91% of all IHC positive (3+) or negative (0/1+) cases. Confirmatory testing of 132 IHC equivocal (2+) cases also yielded highly concordant results between FISH and CISH. CONCLUSIONS: These data validate CISH for the assessment of HER2 gene amplification in breast tumours and, confirm CISH as a valid alternative to FISH in HER2 testing.


Assuntos
Neoplasias da Mama/química , Neoplasias da Mama/genética , Compostos Cromogênicos , Genes erbB-2 , Hibridização in Situ Fluorescente , Hibridização In Situ , Técnicas de Amplificação de Ácido Nucleico , Adulto , Idoso , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Alemanha , Humanos , Imuno-Histoquímica , Hibridização In Situ/métodos , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos Prospectivos , Trastuzumab
9.
Neuron ; 67(1): 75-85, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20624593

RESUMO

HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.


Assuntos
Fenômenos Biofísicos/genética , Canais Iônicos/metabolismo , Ligantes , Animais , Fenômenos Biofísicos/efeitos dos fármacos , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Fluorometria/métodos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Microinjeções/métodos , Oócitos , Técnicas de Patch-Clamp/métodos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Conformação Proteica/efeitos dos fármacos , Fatores de Tempo , Xenopus laevis
10.
Infect Genet Evol ; 9(6): 1381-91, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19800991

RESUMO

Pantoea species are ubiquitous in nature and occasionally associated with infections caused by contaminated clinical material. Hence, Pantoea agglomerans is considered as an opportunistic pathogen of humans. Since species of the genus Pantoea and closely related species of other Enterobacteriaceae genera are phenotypically very similar, many clinical isolates are misassigned into P. agglomerans based on the use of quick commercial-offered biochemical tests. Our objective was to find markers enabling discrimination between clinical and plant isolates and to assess their virulence potential. We characterized 27 Pantoea strains, including 8 P. agglomerans isolates of clinical, and 11 of plant origin by biochemical tests and genotyping, including analysis of 16S rDNA and gapA gene sequences, and pattern polymorphisms of ITS- and ERIC/REP-DNA. All data showed that no discrete evolution occurred between plant-associated and clinical P. agglomerans isolates. Based on the typing results, five clinical- and five plant-associated P. agglomerans strains representing the majority of clades were tested on a model plant and in embryonated eggs. On soybean plants P. agglomerans strains independent of their origin could develop stable epiphytic populations. Surprisingly, in the embryonated egg model there was no difference of virulence between clinical and vegetable P. agglomerans isolates. However, these strains were significantly less virulent than a phytopathogenic P. ananatis isolate. We suggest that, independent of their origin, all P. agglomerans strains might possess indistinguishable virulence potential.


Assuntos
Infecções por Enterobacteriaceae/microbiologia , Glycine max/microbiologia , Pantoea , Doenças das Plantas/microbiologia , Adesinas Bacterianas/análise , Adesinas Bacterianas/genética , Animais , Embrião de Galinha , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Intergênico/análise , DNA Intergênico/genética , Infecções por Enterobacteriaceae/complicações , Infecções por Enterobacteriaceae/diagnóstico , Evolução Molecular , Marcadores Genéticos , Humanos , Infecções Oportunistas/complicações , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/microbiologia , Pantoea/genética , Pantoea/patogenicidade , Filogenia , Polimorfismo Genético , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Virulência
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