Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Combinação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação/efeitos dos fármacos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminofenóis/uso terapêutico , Benzodioxóis/uso terapêutico , Criança , Feminino , Humanos , Indóis/uso terapêutico , Masculino , Pessoa de Meia-Idade , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Pirrolidinas/uso terapêutico , Quinolonas/uso terapêutico , Adulto JovemRESUMO
Anoctamin 1 (ANO1)/TMEM16A is a Cl(-) channel activated by intracellular Ca(2+) mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca(2+). Here, we demonstrate that two helices, "reference" and "Ca(2+) sensor" helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca(2+)-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca(2+)-dependent activation because neutralization of these charges change the Ca(2+) sensitivity. We now predict that the Ca(2+) sensor helix attaches to the reference helix in the resting state, and as intracellular Ca(2+) rises, Ca(2+) acts on the sensor helix, which repels it from the reference helix. This Ca(2+)-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Ativação do Canal Iônico , Anoctamina-1 , Sítios de Ligação , Canais de Cloreto/química , Canais de Cloreto/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Propriedades de Superfície , TransfecçãoRESUMO
Intracellular Ca(2+) signal is a key regulator of axonal growth during brain development. As transient receptor potential (TRP) channels are permeable to Ca(2+) and mediate numerous brain functions, it is conceivable that many TRP channels would regulate neuronal differentiation. We therefore screened TRP channels that are involved in the regulation of neurite growth. Among the TRP channels, the Trpm2 level was inversely associated with neurite growth. TRPM2 was highly expressed in embryonic brain. Pharmacological perturbation or knockdown of TRPM2 markedly increased the axonal growth, whereas its overexpression inhibited the axonal growth. Addition of ADP ribose, an endogenous activator of TRPM2, to PC12 cells significantly repressed the axonal growth. TRPM2 was actively involved in the neuronal retraction induced by cerebrospinal fluid-rich lysophosphatidic acid (LPA). More importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Therefore, we conclude that TRPM2 mediates the LPA-induced suppression of axonal growth, which provides a long-sought mechanism underlying the effect of LPA on neuronal development.
Assuntos
Encéfalo/metabolismo , Neuritos/metabolismo , Neurogênese , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose/farmacologia , Animais , Encéfalo/citologia , Encéfalo/embriologia , Células Cultivadas , Células HEK293 , Humanos , Lisofosfolipídeos/farmacologia , Camundongos , Neuritos/efeitos dos fármacos , Células PC12 , Ratos , Canais de Cátion TRPM/genéticaRESUMO
Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Anoctamina-1 , Cálcio/farmacologia , Canais de Cloreto/química , Canais de Cloreto/deficiência , Canais de Cloreto/genética , Condutividade Elétrica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Transporte de Íons/efeitos dos fármacos , Camundongos , Oócitos/metabolismo , Pilocarpina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Salivação/efeitos dos fármacos , XenopusRESUMO
Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca2+. Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function.
Assuntos
Anoctaminas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Ativação do Canal Iônico , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transdução de Sinais , Animais , Anoctaminas/química , Cálcio/metabolismo , Células HEK293 , Humanos , Intestinos/citologia , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Proteínas de Membrana/química , Camundongos Endogâmicos C57BL , Proteínas de Transferência de Fosfolipídeos/química , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sódio/farmacologiaRESUMO
Cl(-) efflux through Ca(2+)-activated Cl(-) channels (CaCCs) in secretory epithelial cells plays a key role in the regulation of fluid secretion. The fluid and electrolyte secretion is closely related to intracellular pH. CaCCs have been known to be inhibited by intracellular acid. However, the molecular mechanism for the inhibition remains unknown. Anoctamin 1 (ANO1) is a Ca(2+)-activated Cl(-) channel that mediates numerous physiological functions including fluid secretion in secretory epithelia. However, little is known about whether ANO1 can be modulated by change of intracellular pH. Here, we demonstrate that Ca(2+)-induced activation of ANO1 and its homolog ANO2 are strongly inhibited by intracellular acid. Intracellular acid caused a rightward shift of the concentration-response curve of Ca(2+) in activating ANO1 and ANO2. To identify the location of the acid-induced inhibition, mutations were made on each of all histidine residues in cytoplasmic part of ANO1. However, none of the His-mutant showed the reduction in the acid-induced inhibition. Furthermore, mutation on Glu- or Asp-residues in the multiple acidic-amino acid regions was ineffective in blocking the acid-induced inhibition. Because the Ca(2+)-binding site of a fungal anoctamin (nhTMEM16) was uncovered by crystallography, mutagenesis was performed in this region. Surprisingly, mutations at Glu, Asp or Asn residues in the hydrophobic core that are known to be essential for Ca(2+)-induced activation of ANO1 blocked the acid-induced inhibition. These results suggest that protons interfere with Ca(2+) at the Ca(2+) binding site of ANO1. These findings provide a molecular mechanism underlying the acid-induced inhibition of ANO1, which may contribute to control fluid and electrolyte secretion in the secretory epithelia.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Prótons , Anoctamina-1 , Células Cultivadas , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Células HEK293 , Humanos , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.
Assuntos
Cálcio/fisiologia , Canais de Cloreto/metabolismo , Temperatura Alta , Nociceptores/metabolismo , Animais , Anoctamina-1 , Células Cultivadas , Agonistas dos Canais de Cloreto , Canais de Cloreto/deficiência , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Nociceptores/fisiologia , Medição da Dor/métodos , Ratos , Ratos Sprague-DawleyRESUMO
A gold-deposited optical fiber sensor system based on surface plasmon resonance (SPR) was developed for optical measurement of neuronal activity. To enhance the sensitivity of localized SPR and to make a precise and safe contact with the cellular membrane, we designed a tapered optical probe of 1 µm diameter at the tip of the fiber. By wet etching and gold evaporating processes, pencil-shaped optical probes were successfully fabricated. The SPR system with the sharp optical probe was integrated with a conventional patch clamping system to realize a simultaneous optical and electrical recording on a single neuron. Although the shape of optical signal is not clear due to tiny change of intrinsic optical properties on the neuron, optical and electrical signals were simultaneously changed by capsaicin stimulation. Furthermore, our designed fiber probe can be applicable to localized optical stimulation as well as in vivo optical neuroprosthetic devices.
Assuntos
Potenciais de Ação/fisiologia , Tecnologia de Fibra Óptica/instrumentação , Neurônios/fisiologia , Ressonância de Plasmônio de Superfície/instrumentação , Imagens com Corantes Sensíveis à Voltagem/instrumentação , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Superparamagnetic Fe(3)O(4)@SiO(2) core-shell nanoparticles (ca. 30 nm diameter), which are surface-modified with a thiol-specific functional group, exhibit superb capturing efficiency toward cysteinyl peptides without contamination from nonspecifically interacting peptides, as clearly evidenced through LC/MS/MS analysis.