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Expressão em levedura e purificação da Ca2+ATPase do retículo sarcoplasmático de coelho / Purification of rabbit sarcoplamic reticulum Ca2+-ATPase expressed in yeastEduardo Moraes Rego Reis 12 September 2000 (has links)
Este estudo descreve um novo método para a produção da Ca2+-ATPase do retículo sarcoplasmático de coelho em levedura utilizando um vetor de expressão regulado por choque térmico. Após solubilização das membranas de levedura com lisofosfatidilcolina, a introdução de um \"tag\" de 6 histidinas na extremidade amino-terminal da Ca2+-ATPase permitiu a sua purificação por cromatografia de afinidade utilizando uma resina carregada com níquel. Utilizando essa estratégia, foi possível obter frações enriquecidas em até 75% de Ca2+-ATPase recombinante, algo não descrito ainda na literatura. A 6xHis Ca2+-ATPase solubilizada em LPC e purificada em coluna de níquel se mantém estável desde que seja introduzido DOPC juntamente com o detergente nas etapas de lavagem e eluição. Nessas condições, a enzima purificada possui elevada atividade ATPásica cálcio-dependente (1.5-2.0 µmol/mg proteína.min) durante vários minutos de reação. A titulação da atividade ATPásica em função do cálcio livre demonstrou que a 6xHis Ca2+-ATPase purificada possui alta afinidade para o íon (K0.5= 0.15 µlM) e manteve uma forte cooperatividade na ativação por cálcio (nH = 2.07). A quantidade e o grau de pureza obtidos são suficientes para permitir a caracterização bioquímica e espectroscópica de mutantes pontuais da Ca2+-ATPase já construídos e expressos em levedura. A conversão da energia presente em ligações químicas em gradiente eletroquímico é um tema central da bioenergética. Espera-se que o estudo dos mutantes pontuais de triptofano da Ca2+-ATPase gerados nesse trabalho contribua para uma melhor compreensão do mecanismo de acoplamento entre a hidrólise de ATP e o transporte vetorial de íons nesse modêlo de estudo de proteínas de transporte. / We describe in this work a new method for the production of SERCA-l Ca2+-ATPase in yeast using a heat-shock regulated expression vector. Following solubilization of yeast membranes with lysophospholipids, the presence of an hexahistidine tag introduced at the Nterminal end of the Ca2+-ATPase allowed its purification by metal chelating affinity chromatography using a nickel-NTA resin. Using this procedure highly enriched ftactions (75% oftotal protein in the ftaction) of yeast-expressed rabbit Ca2+-ATPase were obtained. Detergent-solubilized 6xHis-Ca2+-ATPase retained highly active (1.5 - 2 µmol/mg protein .min) calcium-dependent, vanadate inhibitable ATPase activity as determined by 32P-γ-ATP hydrolysis. Titration of ATPase activity as a function of ftee calcium revealed high Ca2+ affinity (K0.5 =~ 0.15 µM) and the persistence of a strong cooperative pattem of calcium activation (Hill number of 2.07). The yield and purity of 6xHis Ca2+-ATPase fractions produced with this method allows the biochemical and spectroscopic characterization of Ca2+-ATPase mutants produced in the course of this work. Conversion of the energy present in chemical bonds to electrochemical gradient is a central theme of bioenergetics. It is hoped that the study of the Ca2+-ATPase tryptophan mutants generated in this work will contribute to a better understanding of the coupling mechanism between ATP hydrolysis and the vectorial transport of ions across membranes that occur in this model system.
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Biochemical and biophysical characterization of Ca2+ channel complexes in neurotransmission / 神経伝達に関わるCa2+チャネル複合体の生化学・生物物理学的解明Uriu, Yoshitsugu 24 September 2010 (has links)
Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15675号 / 工博第3333号 / 新制||工||1503(附属図書館) / 28212 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 跡見 晴幸, 教授 濵地 格 / 学位規則第4条第1項該当
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INSULIN ACTIONS ON HIPPOCAMPAL NEURONSMaimaiti, Shaniya 01 January 2017 (has links)
Aging is the main risk factor for cognitive decline. The hippocampus, a brain region critical for learning and memory formation, is especially vulnerable to normal and pathological age-related cognitive decline. Dysregulation of both insulin and intracellular Ca2+ signaling appear to coexist and their compromised actions may synergistically contribute to neuronal dysfunction with aging. This dissertation focused on the interaction between insulin, Ca2+ dysregulation, and cognition in hippocampal neurons by examining the contributions of insulin to Ca2+ signaling events that influence memory formation. I tested the hypothesis that insulin would increase cognition in aged animals by altering Ca2+-dependent physiological mechanisms involved in learning. The possible effects of insulin on learning and memory in young and aged rats were studied. In addition, the effects of insulin on the Ca2+-dependent afterhyperpolarization in CA1 pyramidal hippocampal neurons from young and aged animals were compared. Further, primary hippocampal cultures were used to examine the possible effects of insulin on voltage-gated Ca2+ channel activity and Ca2+-induced Ca2+-release; mechanisms known to influence the AHP.
We found that intranasal insulin improved memory in aged F344 rats. Young and aged F344 rats were treated with Humalog®, a short-acting insulin analog, or Levemir®, a long-acting insulin analog. The aged rats performed similar to young rats in the Morris Water Maze, a hippocampal dependent spatial learning and memory task. Electrophysiological recordings from CA1 hippocampal neurons revealed that insulin reduced the age-related increase in the Ca2+-dependent afterhyperpolarization, a prominent biomarker of brain aging that is associated with cognitive decline. Patch clamping recording from hippocampal cultured neurons showed that insulin reduced Ca2+ channel currents. Intracellular Ca2+ levels were also monitored using Fura-2 in response to cellular depolarization. Results indicated that a reduction in Ca2+-induced Ca2+-release from intracellular stores occurred in the presence of insulin.
These results suggest that increasing brain insulin levels in aged rats may have improved memory by reducing the AHP and intracellular Ca2+concentrations. This study indicates a possible mechanism responsible for the beneficial effects of intranasal insulin on cognitive function absorbed in selective Alzheimer’s patients. Thus, insulin therapy may reduce or prevent age-related compromises to Ca2+ regulatory pathways typically associated with cognitive decline.
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Les voies de signalisation calciques impliquées dans la réponse à l’étirement dans les artères intrapulmonaires. Modifications dans l’hypertension pulmonaire / Ca2+ signaling pathways involved in response to stretch in pulmonary arteries. Implication in pulmonary hypertensionGilbert, Guillaume 29 October 2014 (has links)
L’hypertension pulmonaire (HTP) est la principale pathologie de la circulation pulmonaire. Elle secaractérise par une augmentation maintenue de la pression dans les artères intrapulmonaires (AIP) (> à 25mmHg au repos). Cette pression exerce des forces d’étirement au niveau des cellules musculaires lisses desartères intrapulmonaires (CML d’AIP). Au niveau des CML, des canaux mécanosensibles appelés des SAC(« stretch-activated channels ») permettent de transformer un stimulus mécanique d’étirement en uneréponse biologique de contraction : c’est le tonus myogénique. Le Ca2+ est un second messager cellulairequi peut être aussi bien mobilisé depuis le milieu extracellulaire que depuis les réserves calciquesintracellulaires. Une augmentation de sa concentration cytoplasmique induit la contraction des CML. Grâceà des techniques de patch-clamp, de microspectrofluorimétrie, d’immunomarquages et à une approchepharmacologique, nous avons mis en évidence les voies de signalisations calciques qui sont mises en placeà la suite d’un étirement des CML d’AIP. Les expériences ont été réalisées à la fois chez des rats normaux etsur deux modèles de rats présentant une HTP (rats hypoxique chroniques et rats monocrotalines). Lesrésultats montrent que chez les rats normaux un étirement induit un influx de Ca2+ par les SAC. Cet influxcalcique est amplifié par (1) une hyperpolarisation de la membrane plasmique via l’activation de canauxBKCa, (2) une sortie de Ca2+ par les récepteurs à la ryanodine de type 1 (RyR1) du réticulum sarcoplasmique(RS) sous-membranaire. Afin de rétablir l’homéostasie calcique, les mitochondries tamponnent le Ca2+cytosolique. Chez les rats souffrant d’HTP, l’influx de Ca2+ par les SAC et l’amplification calcique par les RyRsont plus importants. Cette amplification est due à une réorganisation des réserves calciquesintracellulaires, notamment chez les rats monocrotalines. De plus, une association fonctionnelle entre lesréserves calciques du RS et les cavéoles conduit à des réponses calciques plus importantes après unétirement chez les rats HTP. Enfin, nous avons mis en évidence la présence de canaux mécanosensiblesPiezo1 dans les AIP de rats. En conclusion, l’organisation spatiale des partenaires calciques au sein des CMLd’AIP est importante pour la signalisation cellulaire et joue un rôle majeur dans l’HTP. / Pulmonary hypertension (PH) is the main disease of the pulmonary circulation. This pathology ischaracterized by an increase of the intrapulmonary arterial (PA) pressure at rest (> 25 mmHg). This pressureexerts stretch forces on pulmonary arterial smooth muscle cells (PASMC). Stretch-activated channels (SAC)are present in PASMC and are able to transform a mechanical stimulus of stretch into a biological responseof contraction, a phenomenon called myogenic tone. Ca2+ is a second messenger that can be mobilizedfrom both the extracellular medium and intracellular Ca2+ stores. An increase of the intracellular Ca2+concentration ([Ca2+]i) leads to PASMC contraction. Using patch-clamp, microspectrofluorimetry,immunostainings and a pharmacological approach, we highlight Ca2+ signaling pathways induced by stretchin PASMC. Experiments were performed in normal rats and in two models of PH (chronically hypoxic ratsand monocrotaline rats). We showed that in normal rats a stretch induces a Ca2+ influx through SAC whichis amplified by (1) a plasma membrane hyperpolarization by BKCa channels and (2) a Ca2+ amplification bysubplasmalemnal ryanodine receptor 1 (RyR) of the sarcoplasmic reticulum (SR). Besides, mitochondria areinvolved in buffering cytoplasmic Ca2+. In PH rats, the Ca2+ influx by SAC and the Ca2+ release by RyR areenhanced due to a reorganization of intracellular Ca2+ stores. Furthermore, a functional associationbetween SR and caveolae conduce to a much greater amplification of the stretch-induced Ca2+ increase inPH rats. Finally, we showed that the mechanosensitive channel Piezo1 is expressed in PA. To conclude, thespatial organization of Ca2+ stores in PASMC is important for cell signaling and plays a casual role in PH.
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Elucidation of Ca[2+] channel function in higher brain function / Ca[2+]チャネルの脳高次機能における機能の解明Nakao, Akito 24 September 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18594号 / 工博第3955号 / 新制||工||1608(附属図書館) / 31494 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
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Molecular elucidation of the physiological significance of Ca2+ channelsome in neuronal function / 神経機能におけるCa2+チャネルソームの生理的意義の分子解明に関する研究Takada, Yoshinori 24 November 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19376号 / 工博第4121号 / 新制||工||1635(附属図書館) / 32390 / 新制||工||1635 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM
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Elucidation of signal regulation by interacting molecules and proteins of Ca2+ influx channels / Ca2+チャネル相互作用分子によるシグナル伝達制御の解明Sawamura, Seishirou 23 March 2016 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19753号 / 工博第4208号 / 新制||工||1649(附属図書館) / 32789 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
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Étude des interactions dynamiques de la CaMKII avec le cytosquelette du neuroneLabrie-Dion, Étienne. 18 March 2022 (has links)
Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2012-2013. / La formation des connexions entre les neurones durant le développement et la plasticité des connexions synaptiques une fois établies nécessite une fine régulation au niveau du cytosquelette du neurone. Les interactions entre les microtubules et l'actine du cytosquelette sont à l'origine du déplacement et du guidage de l'extrémité de l'axone en croissance, et de récentes évidences suggèrent qu'elles pourraient être importantes dans la réorganisation synaptique. La protéine kinase Ca2+/calmoduline-dépendante II (CaMKII), une des protéines les plus abondantes du cerveau, pourrait être impliquée dans la régulation de ces interactions. Il a été montré que la sous-forme CaMKIIb, exprimée dans le développement et liant l'actine en situation de faible activité, détecte les oscillations calciques dans le cône de croissance et provoque son attraction. Le mécanisme par lequel la CaMKIIb entraîne le virage du cône est cependant inconnu. L'isoforme CaMKIIa, essentielle dans la potentialisation à long-terme de l'épine dendritique, a été observée s'accumulant sous l'épine dendritique lors d'une forte activité locale, où elle pourrait contrôler la livraison locale de cargos destinés à la synapse. Dans le laboratoire du Dr. De Koninck, nous avons observé ces deux formes se lier à des structures ressemblant à des microtubules pendant une forte stimulation. La liaison de la CaMKII aux microtubules pourrait expliquer le mécanisme d'action de la CaMKIIb dans le virage du cône de croissance ainsi que mettre en évidence un nouveau rôle de la CaMKIIa dans l'épine dendritique. Au cours de mes travaux de maîtrise, j'ai observé la CaMKII et le cytosquelette dans des cultures de neurones d'hippocampe de rats en marquant les protéines avec des anticorps ou en transfectant des protéines de fusion fluorescentes. Mes analyses de colocalisation m'ont permis de montrer que la dépolarisation du neurone provoque le déplacement de la CaMKIIb de l'actine vers les microtubules dans le cône de croissance et la localisation de la CaMKIIa aux microtubules, mais pas aux neurofilaments, dans le neurone plus mature. Les études d'inhibition de la CaMKIIb au cours du développement ainsi que l'étude du guidage du cône de croissance n'ont pas donné de résultats probants permettant d'expliquer le rôle du déplacement de la CaMKIIb. Finalement, il est possible que la liaison de la CaMKIIa aux microtubules sous l'épine puisse être impliquée dans les entrées de microtubules dans l'épine et dans la livraison de récepteurs AMPA.
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Epac2 signaling at the β-cell plasma membraneAlenkvist, Ida January 2016 (has links)
Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.
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CALCIUM REGULATION OF CELL-CELL COMMUNICATION AND EXTRACELLULAR SIGNALINGZou, Juan 12 August 2016 (has links)
As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist.
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