RESUMO
BACKGROUND: Molecularly engineered mice are extensively used as models of cardiovascular diseases, yet little is known about sex differences in the electrophysiology of mouse hearts. METHODS AND RESULTS: This study investigated the influence of sex on drug-induced polymorphic ventricular tachycardia (PVT) in Langendorff-perfused male and female mice hearts (n=54) by injecting a bolus of halothane (1.75 mmol/L) in the perfusate while recording ECGs or optical action potentials (APs). There were no statistically significant differences between male and female hearts (n=54) with respect to mean RR (193+/-5 ms), PR (47+/-1 ms), QT intervals (101+/-3 ms), optical AP durations (APD(75)=23.11+/-4.2 ms), dispersion of refractory periods, and conduction velocities (n=5 male and 5 female). Halothane induced PVTs lasting a mean duration of 90 seconds; in female hearts, 55% of PVTs lasted longer than the median, whereas in male hearts 17% exceeded the mean (P<0.05). The total duration of PVTs exposed a marked sex difference, 378+/-144 seconds in female versus 27+/-10 seconds in male hearts (P<0.05). In optically mapped male hearts, halothane reduced APD(75) (17.61+/-1.6 ms) and then elicited VTs (n=6 of 6), but in female hearts, halothane elicited PVTs (n=1 of 6) or arrested the hearts (n=5 of 6). Except for KCNE1, Northern blots (KCNQ1, MERG, Kv1.5, connexins 40 and 43, TREK1, and TASK1) did not detect sex differences. CONCLUSIONS: This mouse model reveals sex difference in response to a pharmacological challenge yet does not display sex differences in standard electrophysiological parameters. Differences in KCNE1 may contribute to sex differences uncovered by halothane.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Fatores Sexuais , Taquicardia Ventricular/etiologia , Potenciais de Ação , Animais , Mapeamento Potencial de Superfície Corporal , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Halotano , Coração/fisiopatologia , Cinética , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Canais de Potássio/biossíntese , Canais de Potássio/genética , RNA Mensageiro/biossíntese , Período Refratário Eletrofisiológico , Taquicardia Ventricular/induzido quimicamente , Taquicardia Ventricular/fisiopatologiaRESUMO
Prostaglandins (PGs) are important mediators of bone response to growth factors, hormones, inflammation, or mechanical strains. In this study, we show that in MG63 osteosarcoma cells, prostaglandin E2 (PGE2) produces the opening of a large conductance Ca2+-dependent K+ channel (BK). This PGE2-mediated channel opening induces the recruitment of various tyrosine-phosphorylated proteins on the hSlo alpha-subunit of BK. Because the C-terminal domain of hSlo encompasses an immunoreceptor tyrosine-based activation motif (ITAM), we show that the Syk nonreceptor tyrosine kinase, reported yet to be expressed mainly in hematopoietic cells, is expressed also in osteoblastic cells, and recruited on this ITAM after a PGE2-induced docking/activation process. We show that Syk/hSlo association is dependent of an upstream Src-related tyrosine kinase activity, in accord with the classical two-step model described for immune receptors. Finally, we provide evidence that this Syk/hSlo interaction does not affect the electrical features of BK channels in osteosarcoma cells. With these data, we would like to suggest the new notion that besides its conductance function, hSlo channel can behave in bone cells, as a true transduction protein intervening in the bone remodeling induced by PGE2.
Assuntos
Dinoprostona/farmacologia , Precursores Enzimáticos/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Células COS , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Dados de Sequência Molecular , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/genética , Transdução de Sinais , Quinase Syk , Células Tumorais Cultivadas , Quinases da Família src/metabolismoRESUMO
UNLABELLED: Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. INTRODUCTION: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. METHODS: Interaction of FAK with the C terminus of the hSlo alpha-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. RESULTS: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. CONCLUSIONS: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.
Assuntos
Cálcio/metabolismo , Osteoblastos/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem da Célula , Eletrofisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Osteoblastos/patologia , Plasmídeos/metabolismo , Canais de Potássio/química , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
The TREK-1 channel is a temperature-sensitive, osmosensitive and mechano-gated K+ channel with a regulation by Gs and Gq coupled receptors. This paper demonstrates that TREK-1 qualifies as one of the molecular sensors involved in pain perception. TREK-1 is highly expressed in small sensory neurons, is present in both peptidergic and nonpeptidergic neurons and is extensively colocalized with TRPV1, the capsaicin-activated nonselective ion channel. Mice with a disrupted TREK-1 gene are more sensitive to painful heat sensations near the threshold between anoxious warmth and painful heat. This phenotype is associated with the primary sensory neuron, as polymodal C-fibers were found to be more sensitive to heat in single fiber experiments. Knockout animals are more sensitive to low threshold mechanical stimuli and display an increased thermal and mechanical hyperalgesia in conditions of inflammation. They display a largely decreased pain response induced by osmotic changes particularly in prostaglandin E2-sensitized animals. TREK-1 appears as an important ion channel for polymodal pain perception and as an attractive target for the development of new analgesics.
Assuntos
Nociceptores/metabolismo , Dor/metabolismo , Percepção/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Gânglios Espinais/citologia , Hibridização In Situ , Camundongos , Camundongos Knockout , Fibras Nervosas Amielínicas/metabolismo , Neurônios Aferentes/citologia , Neurônios Aferentes/metabolismo , Medição da Dor , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/genética , RNA Mensageiro/metabolismoRESUMO
The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few available bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an alpha-glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polarized MDCK, where it is anchored by glycosylphosphatidylinositol (GPI) and displays alpha-glucosidase activity. Bin bound to transfected cells and induced a non-specific current presumably related to the opening of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time-dependent appearance of intracytoplasmic vacuoles but did not drive cell lysis. Thus, the dual functionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.
Assuntos
Toxinas Bacterianas/metabolismo , Células Epiteliais/ultraestrutura , Glicosilfosfatidilinositóis/metabolismo , alfa-Glucosidases/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Linhagem Celular , Permeabilidade da Membrana Celular , Culex , Cães , Células Epiteliais/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/ultraestrutura , Controle de Mosquitos , Ensaio Radioligante , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestrutura , alfa-Glucosidases/genéticaRESUMO
Group I metabotropic glutamate receptors (mGluRs) are implicated in diverse processes such as learning, memory, epilepsy, pain and neuronal death. By inhibiting background K(+) channels, group I mGluRs mediate slow and long-lasting excitation. The main neuronal representatives of this K(+) channel family (K(2P) or KCNK) are TASK and TREK. Here, we show that in cerebellar granule cells and in heterologous expression systems, activation of group I mGluRs inhibits TASK and TREK channels. D-myo-inositol-1,4,5-triphosphate and phosphatidyl-4,5-inositol-biphosphate depletion are involved in TASK channel inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels. Mechanisms described here with group I mGluRs will also probably stand for many other receptors of hormones and neurotransmitters.
Assuntos
Cerebelo/fisiologia , Neurônios/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Sítios de Ligação , Células COS , Cálcio/fisiologia , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/fisiologia , Células Cultivadas , Chlorocebus aethiops , Cinética , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Ratos , Ratos Wistar , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , TransfecçãoRESUMO
TASK-1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage- and Ca2+-sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK-1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C-terminal amino acids, Ser-Ser-Val, in TASK-1. Using series of C-terminal TASK-1 deletion mutants and several TASK-1-GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK-1 to the plasma membrane. p11 association with the TASK-1 channel masks an endoplasmic reticulum retention signal identified as Lys-Arg-Arg that precedes the Ser-Ser-Val sequence.