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1.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281197

RESUMO

Psoriasis, a long-lasting and multifactorial skin disease, is related to comorbidities such as metabolic disease, depression, and psoriatic arthritis. Psoriasis occurs due to a variety of factors including keratinocyte hyperproliferation, inflammation, and abnormal differentiation. Proinflammatory cytokines upregulated by increased activation of keratinocytes and immune cells in the skin trigger progression of psoriasis. This study aimed to investigate the effects of anoctamin1 (ANO1) on psoriasis development in vitro and in vivo. We analyzed the proliferation of HaCaT keratinocytes and ANO1-related ERK and AKT signaling pathways after ANO1 inhibitor (T16Ainh-A01 and Ani9) treatment and knock-down of ANO1. Furthermore, after applying imiquimod (IMQ) cream or coapplying IMQ cream and T16Ainh-A01 on mouse ears, we not only observed psoriatic symptoms, including ear thickening, but also quantified the effects of treatment on ERK and AKT signaling-involved proteins and proinflammatory cytokines. Inhibition of ANO1 attenuated the proliferation of HaCaT cells and induced reduction of pERK1/2. Coapplication of IMQ and T16Ainh-A01 on ears of mice reduced not only symptoms of IMQ-induced psoriasis such as thickening and erythema, but also expression of ANO1 and pERK1/2 compared to that of application of IMQ alone. In addition, the expression levels of IL-17A, IL-17F, IL-22, IL-23, IL-6, IL-1ß, and TNF-α increased after applying IMQ and were significantly reduced by coapplying IMQ and T16Ainh-A01. These results aid in understanding the underlying mechanisms of ANO1 in epidermal layer keratinocyte hyperproliferation and suggest the potential of ANO1 as a target to treat psoriasis.


Assuntos
Anoctamina-1/farmacologia , Queratinócitos/efeitos dos fármacos , Psoríase/induzido quimicamente , Acetamidas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células HaCaT , Humanos , Hidrazonas/farmacologia , Imiquimode/efeitos adversos , Imiquimode/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Interleucinas/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos BALB C , Psoríase/metabolismo , Psoríase/patologia , Pirimidinas/farmacologia , Tiazóis/farmacologia
2.
Molecules ; 25(21)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172169

RESUMO

Pyrimidine is a privileged scaffold in many synthetic compounds exhibiting diverse pharmacological activities, and is used for therapeutic applications in a broad spectrum of human diseases. In this study, we prepared a small set of pyrimidine libraries based on the structure of two hit compounds that were identified through the screening of an in-house library in order to identify an inhibitor of anoctamin 1 (ANO1). ANO1 is amplified in various types of human malignant tumors, such as head and neck, parathyroid, and gastrointestinal stromal tumors, as well as in breast, lung, and prostate cancers. After initial screening and further structure optimization, we identified Aa3 as a dose-dependent ANO1 blocker. This compound exhibited more potent anti-cancer activity in the NCI-H460 cell line, expressing high levels of ANO1 compared with that in A549 cells that express low levels of ANO1. Our results open a new direction for the development of small-molecule ANO1 blockers composed of a pyrimidine scaffold and a nitrogen-containing heterocyclic moiety, with drug-like properties.


Assuntos
Anoctamina-1/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Pirimidinas/química , Animais , Anoctamina-1/metabolismo , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Proteínas de Neoplasias/metabolismo , Pirimidinas/farmacologia , Ratos
3.
Exp Neurobiol ; 28(6): 658-669, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31902154

RESUMO

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca2+ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca2+ binding. Because the Ca2+ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca2+ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca2+ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca2+-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca2+ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca2+. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca2+ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca2+ and modulate the activation by Ca2+ and voltage.

4.
Physiol Behav ; 188: 188-193, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29432788

RESUMO

Itch is an unpleasant sensation that evokes behavioral responses such as scratching the skin. Interestingly, it is conceived that the perception of itch sensation is influenced by age. Indeed, accumulating evidence supports the idea that even children or younger adults show distinctive itch sensation depending on age. This evidence implies the presence of a mechanism that regulates the perception of itch sensation in an age-dependent fashion. Therefore, the purpose of the present study was to investigate a putative mechanism for the age-dependent perception of itch sensation by comparing histamine-induced scratching behaviors in 45-day old (D45) and 75-day old male "young adult" mice. The results indicated that, following histamine administration, the D75 mice spent a longer time scratching than D45 mice. However, the intensity of the calcium influx induced by histamine in primary culture of dorsal root ganglia (DRG) neurons was not different between D45 and D75 mice. Moreover, no apparent difference was observed in mRNA levels of a characteristic His-related receptor and ion channel. In contrast, the mRNA levels of Toll-Like Receptor 4 (TLR4) were increased approximately by two-fold in D75 DRG compared with D45 DRG. Additionally, D75-derived DRG neurons exhibited enhanced intracellular calcium increase by lipopolysaccharide (LPS, a TLR4 agonist) than those of D45 mice. Furthermore, intensities of calcium influx induced by histamine were significantly potentiated when co-treated with LPS in D75 DRG neurons, but not in those of D45 mice. Thus, it appears that D75 mice showed enhanced histamine-induced scratching behaviors not by increased expression levels of histamine-related genes, but probably due to augmented TLR4 expression in DRG neurons. Consequently, the current study found that different perception levels of histamine-induced itch sensation are present in different age groups of young adult mice.


Assuntos
Agonistas dos Receptores Histamínicos/toxicidade , Histamina/toxicidade , Percepção/efeitos dos fármacos , Prurido/induzido quimicamente , Prurido/fisiopatologia , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Gânglios Espinais/citologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Percepção/fisiologia , Prurido/patologia , RNA Mensageiro/metabolismo , Receptores Histamínicos/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
5.
Mol Neurobiol ; 55(8): 6589-6600, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29327205

RESUMO

Pain is a protective mechanism that enables us to avoid potentially harmful environments. However, when pathologically persisted and aggravated under severely injured or inflamed conditions, pain often reduces the quality of life and thus is considered as a disease to eliminate. Inflammatory and/or neuropathic mechanisms may exaggerate interactions between damaged tissues and neural pathways for pain mediation. Similar mechanisms also promote the communication among cellular participants in synapses at spinal or higher levels, which may amplify nociceptive firing and subsequent signal transmission, deteriorating the pain sensation. In this pathology, important cellular players are afferent sensory neurons, peripheral immune cells, and spinal glial cells. Arising from damage of injury, overloaded interstitial and intracellular reactive oxygen species (ROS) and intracellular Ca2+ are key messengers in the development and maintenance of pathologic pain. Thus, an ROS-sensitive and Ca2+-permeable ion channel that is highly expressed in the participant cells might play a critical role in the pathogenesis. Transient receptor potential melastatin subtype 2 (TRPM2) is the unique molecule that satisfies all of the requirements: the sensitivity, permeability, and its expressing cells. Notable progress in delineating the role of TRPM2 in pain has been achieved during the past decade. In the present review, we summarize the important findings in the key cellular components that are involved in pathologic pain. This overview will help to understand TRPM2-mediated pain mechanisms and speculate therapeutic strategies by utilizing this updated information.


Assuntos
Nociceptividade , Dor/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Humanos , Inflamação/patologia , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo
6.
BMB Rep ; 51(5): 236-241, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29335069

RESUMO

Anoctamin 1 (ANO1) is an anion channel that is activated by changes in cytosolic Ca2+ concentration and noxious heat. Although the critical roles of ANO1 have been elucidated in various cell types, the control of its gating mechanisms by Ca2+ and heat remain more elusive. To investigate critical amino acid residues for modulation of Ca2+ and heat sensing, we constructed a randomized mutant library for ANO1. Among 695 random mutants, reduced Ca2+ sensitivity was observed in two mutants (mutant 84 and 87). Consequently, the E143A mutant showed reduced sensitivity to Ca2+ but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both Ca2+ and noxious heat. These results suggest that the glutamic acids (E) at 143 and 705 residues in ANO1 are critical for modulation of Ca2+ and/or heat responses. Furthermore, these findings help to provide a better understanding of the Ca2+-mediated activation and heat-sensing mechanism of ANO1. [BMB Reports 2018; 51(5): 236-241].


Assuntos
Anoctaminas/química , Anoctaminas/metabolismo , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Temperatura Alta , Nociceptividade , Sequência de Aminoácidos , Animais , Anoctaminas/genética , Ácido Glutâmico/genética , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Modelos Moleculares , Mutação/genética , Domínios Proteicos , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade
7.
Biochem Biophys Res Commun ; 495(1): 1208-1213, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29183725

RESUMO

Harmful maternal behaviors, such as drinking and smoking, negatively affect embryonic development. In contrast, regular maternal exercise is believed to be beneficial to the fetus. Although it is not surprising that voluntary physical activities are advantageous to fetal development, it remains unclear whether involuntary maternal exercise has similar effects. The constituents of the amniotic fluid (AF) inevitably reflect the maternal plasma. Therefore, it is speculated that exercise-induced changes in maternal plasma can influence fetal development through changes in AF composition. Therefore, we investigated the effect of AF on fetal neurodevelopment and changes in AF composition after involuntary swimming exercise (SE) in an animal model. We found that there was a severe reduction in the number of embryos implanted in the uterus of SE rats. Surprisingly, cortisol level (an inducible stress hormone) was significantly increased in AF from SE rats. In contrast, the growth factors NGF and VEGF were reduced in the AF from SE rats. In the cultured embryonic cortical neurons, the treatment of control (CTL) rat-derived AF significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated signaling that is essential for fetal neurodevelopment. However, the AF extracted from SE rats reversely suppressed the phosphorylation of ERK1/2-mediated signaling in cortical neurons compared to that in CTL rats. Indeed, the co-treatment with control AF and dexamethasone, a synthetic glucocorticoid, inhibited the phosphorylation of ERK1/2 in a dose-dependent manner. This finding suggests that the inhibition of ERK1/2 can be attributed to increased cortisol level in AF resulting from involuntary exercise. Therefore, involuntary maternal swimming increases cortisol level in AF, which ultimately hinders the ERK1/2 signaling pathway in embryonic neurons. These findings also suggest that involuntary maternal exercise can have undesirable effects on fetal neurodevelopment, which is potentially mediated by elevated AF cortisol level.


Assuntos
Líquido Amniótico/metabolismo , Desenvolvimento Embrionário/fisiologia , Hidrocortisona/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neurônios/metabolismo , Prenhez/metabolismo , Estresse Fisiológico/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Condicionamento Físico Animal/métodos , Gravidez , Ratos , Ratos Sprague-Dawley , Natação , Volição/fisiologia
8.
Pflugers Arch ; 467(8): 1677-87, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25231974

RESUMO

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 , Transfecção
9.
Mol Pain ; 10: 5, 2014 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-24450308

RESUMO

BACKGROUND: Various pathological conditions such as inflammation or injury can evoke pain hypersensitivity. That represents the response to innocuous stimuli or exaggerated response to noxious stimuli. The molecular mechanism based on the pain hypersensitivity is associated with changes in many of ion channels in dorsal-root ganglion (DRG) neurons. Anoctamin 1 (ANO1/TMEM16A), a Ca2+ activated chloride channel is highly visible in small DRG neurons and responds to heat. Mice with an abolished function of ANO1 in DRG neurons demonstrated attenuated pain-like behaviors when exposed to noxious heat, suggesting a role in acute thermal nociception. In this study, we further examined the function of ANO1 in mediating inflammation- or injury-induced hyperalgesia or allodynia. RESULTS: Using Advillin/Ano1fl/fl (Adv/Ano1fl/fl) mice that have a functional ablation of Ano1 mainly in DRG neurons, we were able to determine its role in mediating thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury. The thermal hyperalgesia and mechanical allodynia induced by carrageenan injection and spared-nerve injury were significantly reduced in Adv/Ano1fl/fl mice. In addition, flinching or licking behavior after bradykinin or formalin injection was also significantly reduced in Adv/Ano1fl/fl mice. Since pathological conditions augment nociceptive behaviors, we expected ANO1's contribution to the excitability of DRG neurons. Indeed, the application of inflammatory mediators reduced the threshold for action potential (rheobase) or time for induction of the first action potential in DRG neurons isolated from control (Ano1fl/fl) mice. These parameters for neuronal excitability induced by inflammatory mediators were not changed in Adv/Ano1fl/fl mice, suggesting an active contribution of ANO1 in augmenting the neuronal excitability. CONCLUSIONS: In addition to ANO1's role in mediating acute thermal pain as a heat sensor, ANO1 is also capable of augmenting the excitability of DRG neurons under inflammatory or neuropathic conditions and thereby aggravates inflammation- or tissue injury-induced pathological pain.


Assuntos
Canais de Cloreto/metabolismo , Hipersensibilidade/etiologia , Inflamação/complicações , Inflamação/patologia , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Animais , Anoctamina-1 , Bradicinina/farmacologia , Formaldeído/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/etiologia , Hiperalgesia/genética , Hiperalgesia/patologia , Hipersensibilidade/genética , Hipersensibilidade/patologia , Inflamação/genética , Camundongos , Camundongos Knockout , Nociceptividade/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia
10.
Arch Pharm Res ; 35(10): 1823-30, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23139135

RESUMO

The transient receptor potential (TRP) channel superfamily is a set of channel genes that mediate numerous physiological functions such as sensing irritants or detecting temperature changes. Despite their functions, expressional information on TRP channels in various organs is largely elusive. Therefore, we conducted a systematic quantitative comparison of each mRNA expression level of 22 mouse TRP channels in various organs. As a result, we found that average levels of TRP channel transcripts were very low reaching ∼3% of the GAPDH transcript level. Among 22 TRP channels, TRPC1 and TRPM7 were most abundant in the majority of organs. In contrast, TRPV3, TRPV5, TRPV6, TRPC7, TRPM1, and TRPM5 elicited very low message profiles throughout the major organs. Consistent with their functions as molecular sensors for irritants and temperature changes, TRPV1, TRPM8 and TRPA1 showed exclusive expression in sensory ganglia. TRPC3 and TRPM3 were abundant in the sensory ganglia and brain. High levels of transcripts of TRPV2, TRPC6, TRPM4, and TRPM6 were observed in the lung. In addition, channel transcript levels were very low except TRPM7 in the liver. In summary, the expression profile of TRP channels in major tissues provides insight to their physiological functions and therefore application to new drug development.


Assuntos
Perfilação da Expressão Gênica , Expressão Gênica , Canais de Potencial de Receptor Transitório/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Especificidade de Órgãos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
11.
Nat Neurosci ; 15(7): 1015-21, 2012 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-22634729

RESUMO

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-Dawley
12.
Cell Calcium ; 48(4): 202-8, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20934218

RESUMO

Exposure to low temperatures often causes allergic responses or urticaria. Similarly, menthol, a common food additive is also known to cause urticaria, asthma, and rhinitis. However, despite the obvious clinical implications, the molecular mechanisms responsible for inducing allergic responses to low temperatures and menthol have not been determined. Because a non-selective cation channel, transient receptor potential subtype M8 (TRPM8) is activated by cold and menthol, we hypothesized that this channel mediates cold- and menthol-induced histamine release in mast cells. Here, we report that TRPM8 is expressed in the basophilic leukemia mast cell line, RBL-2H3, and that exposure to menthol or low temperatures induced Ca(2+) influx in RBL-2H3 cells, which was reversed by a TRPM8 blocker. Furthermore, menthol, a TRPM8 agonist, induced the dose-dependent release of histamine from RBL-2H3 cells. When TRPM8 transcripts were reduced by siRNA (small interfering RNA), menthol- and cold-induced Ca(2+) influx and histamine release were significantly reduced. In addition, subcutaneous injection of menthol evoked scratching, a typical histamine-induced response which was reversed by a TRPM8 blocker. Thus, our findings indicate that TRPM8 mediates the menthol- and cold-induced allergic responses of mast cells, and suggest that TRPM8 antagonists be viewed as potential treatments for cold- and menthol-induced allergies.


Assuntos
Cálcio/metabolismo , Temperatura Baixa , Liberação de Histamina , Mastócitos/imunologia , Mentol/toxicidade , Canais de Cátion TRPM/metabolismo , Compostos de Anilina/farmacologia , Animais , Comportamento Animal , Linhagem Celular Tumoral , Corantes Fluorescentes/farmacologia , Hipersensibilidade/etiologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Xantenos/farmacologia
13.
Nature ; 455(7217): 1210-5, 2008 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-18724360

RESUMO

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 , Xenopus
14.
J Neurosci ; 26(9): 2403-12, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16510717

RESUMO

TRPV1, a cloned capsaicin receptor, is a molecular sensor for detecting adverse stimuli and a key element for inflammatory nociception and represents biophysical properties of native channel. However, there seems to be a marked difference between TRPV1 and native capsaicin receptors in the pharmacological response profiles to vanilloids or acid. One plausible explanation for this overt discrepancy is the presence of regulatory proteins associated with TRPV1. Here, we identify Fas-associated factor 1 (FAF1) as a regulatory factor, which is coexpressed with and binds to TRPV1 in sensory neurons. When expressed heterologously, FAF1 reduces the responses of TRPV1 to capsaicin, acid, and heat, to the pharmacological level of native capsaicin receptor in sensory neurons. Furthermore, silencing FAF1 by RNA interference augments capsaicin-sensitive current in native sensory neurons. We therefore conclude that FAF1 forms an integral component of the vanilloid receptor complex and that it constitutively modulates the sensitivity of TRPV1 to various noxious stimuli in sensory neurons.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neurônios Aferentes/fisiologia , Canais de Cátion TRPV/fisiologia , Ácidos/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose , Biotinilação/métodos , Western Blotting/métodos , Western Blotting/estatística & dados numéricos , Capsaicina/farmacologia , Células Cultivadas , Clonagem Molecular/métodos , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Mutação , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Estrutura Terciária de Proteína/fisiologia , RNA Interferente Pequeno/farmacologia , Ensaio Radioligante/métodos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Temperatura , Transfecção/métodos , Ubiquitina/metabolismo
15.
Eur J Neurosci ; 17(12): 2630-8, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12823470

RESUMO

Ion channels in sensory neurons are molecular sensors that detect external stimuli and transduce them to neuronal signals. Although Ca2+-activated nonselective cation (CAN) channels were found in many cell types, CAN channels in mammalian sensory neurons are not yet identified. In the present study, we describe an ion channel that is activated by intracellular Ca2+ in cultured rat sensory neurons. Half-maximal concentration of Ca2+ in activating the CAN channel was approximately 780 micro m. The current-voltage relationship of this channel was linear with a unit conductance of 28.8 +/- 0.4 pS at -60 mV in symmetrical 140 mm Na+ solution. The CAN channel was permeable to monovalent cations such as Na+, K+, Cs+, and Li+, but poorly permeable to Ca2+. The CAN channel in mammalian sensory neurons was reversibly blocked by intracellular adenine nucleotides, such as ATP, ADP, and AMP. Interestingly, single-channel currents activated by Ca2+ were blocked by fenamates, such as flufenamic acid, a class of nonsteroidal anti-inflammatory drugs. Thus, these results suggest that CAN channels in mammalian sensory neurons would participate in modulating nociceptive neural transmission in response to ever-changing intracellular Ca2+ in the local microenvironment.


Assuntos
Cálcio/metabolismo , Canais Iônicos/fisiologia , Neurônios Aferentes/fisiologia , Nucleotídeos de Adenina/farmacologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios não Esteroides/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Capsaicina/farmacologia , Células Cultivadas , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ácido Egtázico/farmacologia , Condutividade Elétrica , Espaço Extracelular/metabolismo , Gânglios Espinais , Ibuprofeno/farmacologia , Potenciais da Membrana/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Receptores de Droga/fisiologia , ortoaminobenzoatos/farmacologia
16.
Protein Expr Purif ; 25(1): 149-59, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12071710

RESUMO

The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics. ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin. In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification. Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h. Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95%. To examine the function of ErmSF in vivo and in vitro, its activity in E. coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B. subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated. The ErmSF in E. coli conferred resistance to erythromycin, whereas E. coli harboring an empty vector, pET23b, was susceptible. The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins. However, the truncated substrates were methylated with decreased efficiencies. Almost all of domain V was monomethylated with less than 0.2 pM S-[methyl-(3)H]adenosylmethionine concentration. The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition.


Assuntos
Antibacterianos/farmacologia , Resistência a Medicamentos , Histidina/farmacologia , Macrolídeos , Metiltransferases/química , Metiltransferases/isolamento & purificação , Estreptogramina B/farmacologia , Bacillus subtilis/metabolismo , Cromatografia em Camada Delgada , Clonagem Molecular , Metilação de DNA , Escherichia coli/metabolismo , Vetores Genéticos , Cinética , Lincosamidas , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura
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