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1
Adney, Scott. "Protein Kinase C Dependent Inhibition of Kir3.2 (GIRK2) Channel Activity and Its Molecular Determinants." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3214.
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Inwardly rectifying potassium (Kir) channels are critically important for regulating resting membrane potential in excitable cells, a job underscored by the severe pathophysiology associated with channel dysfunction. While all Kir channels require the activating lipid PIP2, many of these channels have diverse modulatory factors that couple to PIP2-dependent gating. Channels in the Kir3 (GIRK) family, in particular, have several co-activating elements, including G-protein betagamma subunits, ethanol, and sodium. During stimulation of Gq-coupled receptors, downstream activation of Protein Kinase C can phosphorylate and inhibit Kir3 channels, yet the mechanism of inhibition and phosphorylation sites are incompletely understood. We took a combined experimental and computational approach using neuronal Kir3.2 to investigate how phosphorylation at a putative PKC site identified in Kir3.1/3.4 could lead to channel inhibition. Kir3.2 inhibition was found to depend on the phosphorylation state of Ser-196, although mutagenesis data suggest it functions as an allosteric regulator of PKC inhibition. MD simulations identified a molecular switch whereby phosphorylation of Ser-196 recruits a critical gating residue, Arg-201, away from the sodium coordination site Asp-228. Neutralization of Ser-196 or Arg-201 resulted in less active channels which exhibited increased sensitivity to PKC inhibition. Additionally the interplay of PIP2 and PKC inhibition was examined in depth using homomeric Kir3.2, revealing that increases in channel-PIP2 interactions limit sensitivity to PKC inhibition, whereas low levels of PIP2 increase PKC sensitivity. Neutralization of Ser-196 uncoupled PKC inhibition from this PIP2 dependence. These studies suggest a model whereby PKC inhibition can occur along PIP2-dependent and PIP2-independent pathways, depending on the phosphorylation state of Ser-196.
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Schmidt, Claas [Verfasser], and Horst [Akademischer Betreuer] Lemoine. "Charakterisierung neuartiger K(ATP)-Kanalöffner vom Benzothiadiazin-, Benzofuran-, Benzothiophen- und Benzothiazol-Typ an HEK 293-(SUR2B/Kir6.1)- und CHO-(SUR1/Kir6.2)-Zellen - Funktioneller Nachweis einer dritten Bindungsstelle für Benzothiadiazin-Derivate am (SUR2B/Kir6.1)-K(ATP)-Kanal / Claas Schmidt. Gutachter: Horst Lemoine." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/1071947117/34.
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Měsíčková, Klára. "Elektrofyziologická charakterizace membránového kanálu Kir2.1." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2018. http://www.nusl.cz/ntk/nusl-377661.
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The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.
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Wang, Runping. "Mechanistic Insight into Subunit Stoichiometry for KIR Channel Gating: Ligand Binding, Gating, Binding-Gating Coupling, Coordination, and Cooperativity." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/27.
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Ligand-gated ion channels couple intra- and extracellular chemical signals to cellular excitability. In response to a specific ligand, these channels change their permeability to certain ions by opening or closing their ion conductive pathway, a controlling mechanism known as channel gating. Although recent studies with X-ray crystallography and site-directed mutagenesis have revealed several structures potentially important for channel gating, the gating mechanism is still elusive. Ligand-dependent channel gating involves a series of transient events and asymmetric movements of individual subunits. Understanding of these events appears to be a challenge to current approaches in gating studies by using the homomeric wild-type or mutant channels. I therefore took an alternative approach by constructing heteromeric channels. Subunit stoichiometric studies of the Kir1.1 channel showed that a minimum of one functional subunit was required for the pH-dependent gating of the channel. Four subunits in this channel were coordinated as dynamic functional dimers. In Kir6.2 channel, stoichiometry for proton-binding was almost identical to that for channel gating in the M2 helix, suggesting a one-to-one direct coupling of proton binding in C-terminus to channel gating in M2 helix. Positive cooperativity was suggested among subunits in both the proton binding and channel gating. Ligand binding can be differentiated from channel gating by studying the ATP-dependent gating of Kir6.2 channel. Disruptions in ATP binding were found to change both the potency and efficacy of the concentration-dependent curves, while the baseline activity instead of maximum inhibition was affected by disruptions of channel gating. Four subunits in the Kir6.2 channel undergo negative cooperativity in ATP binding and positive cooperativity in channel gating. The ligand binding was coupled to the gating mechanism in the same subunit and neighboring subunits, although the intrasubunit coupling was more effective. These results are well described with the operational model which we have applied to ion channel studies for the first time. By manipulating the relative distance and the interaction of two transmembrane helices, the inner helix bundle of crossing was found to not only serve as a gate but also determine the consequence of ligand binding.
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Mankouri, Jamel. "Genetic mutations in the Kir6.2 subunit of KATP channels which cause CHI." Thesis, University of Leeds, 2006. http://etheses.whiterose.ac.uk/333/.
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The role of KATP channels have been best characterized in the pancreas where they are crucial for the regulation of insulin secretion from pancreatic ?-cells. In the open state, KATP channels maintain the ?-cell membrane resting potential inhibiting insulin secretion. In response to elevated glucose levels KATP channels close mediating membrane depolarization, opening of voltage-gated Ca 2+ channels, and subsequent insulin secretion. KATP channel current is controlled both by the metabolic regulation of the channel mediated via changes in the [ATP/ADP] ratio and the cell surface density of channels. Using functional and cell biological approaches the current study identifies three trafficking motifs in the Kir6.2 subunit controlling the surface density of KATP channels each with an associated genetic disorder. The first, a di-acidic motif (280DXE282) was found to mediate efficient export of the channels from the endoplasmic reticulum. A mutation in this motif,(E282K), which causes congenital hyperinsulinism, inhibited this export thereby reducing surface channel density. The second, a tyrosine-based motif (330YSKF333) was found to be necessary for clathrin-mediated internalization of the surface expressed channels. Disruption of this motif through mutations (Y330C & F3331)causing permanent neonatal diabetes mellitus inhibited internalization, enhancing surface channel density. Finally, an acidic di-leucine motif (352DRSLL356) was found to regulate rapid recycling of internalized KATP channels. Disruption of this motif through a non-insulin dependent diabetes mellitus causing mutation (L355P) enhanced recycling and channel surface expression. In summary, the current study identified motifs that control biosynthetic and endocytotic trafficking pathways and thereby the cell surf?ce expression of pancreatic KATP channels. More importantly, the study reports that genetic mutations can disrupt each of these motifs and alter the channel cell surface density, thereby causing disease. Mutations that prevent biosynthetic export cause congenital hyperinsulinism whereas mutations that inhibit internalisation or enhance recycling cause diabetes mellitus. The study thus provides a link between genetic mutations, trafficking defects and disorders of insulin secretion.
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Schulz, Miriam. "Untersuchungen zur Interaktion aktivierender und inhibitorischer Nukleotide bei der Regulation von SUR1-Kir6.2-Kanälen." [S.l. : s.n.], 2005. http://www.gbv.de/dms/bs/toc/500892156.pdf.
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Mountadem, Sarah. "Contribution des canaux astrocytaires Kir4.1 dans l'allodynie mécanique." Thesis, Université Clermont Auvergne (2017-2020), 2020. http://www.theses.fr/2020CLFAC060.
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La douleur inflammatoire chronique est une affection fréquente et invalidante qui est maintenue par une sensibilisation centrale, responsable de l’apparition de l’hypersensibilité douloureuse. Alors que la réaction microgliale contribue à l'apparition de la douleur chronique, l’activation des astrocytes et des médiateurs associés semble jouer un rôle essentiel dans le maintien de la douleur chronique et de la sensibilisation centrale. Les canaux potassiques entrants rectifiants Kir4.1 sont majoritairement exprimés par les astrocytes et sont impliqués dans les fonctions physiologiques astrocytaires, en particulier en contrôlant leur potentiel de membrane au repos. Ils interviennent aussi dans les interactions astrocytes-neurones en contrôlant la recapture du glutamate et la concentration extracellulaire de potassium (K+). De plus, il a été démontré que le dysfonctionnement de ces canaux est associé à plusieurs pathologies du système nerveux central, notamment l'épilepsie, la maladie de Huntington, la dépression ou la sclérose amyotrophique latérale. Cependant, le rôle du canal Kir4.1 central dans la douleur n'a pas été clairement étudié. Dans ce projet, nous avons exploré l'implication des canaux Kir4.1 astrocytaires du sous-noyau caudal (Sp5C) du trijumeau dans la chronicisation de l'hypersensibilité à la douleur inflammatoire trigéminale. En combinant des techniques biochimiques, électrophysiologiques et comportementales, nous avons montré chez le rat que l’allodynie mécanique provoquée par l’injection sous-cutanée d’une subtance algogène (Adjuvant Complet de Freund, CFA) est associée à une activation des astrocytes, qui se traduit par des changements de leur morphologie et de leurs propriétés intrinsèques, dont une diminution drastique des courants Kir4.1. Cette diminution est corrélée à une baisse de l’expression protéique des canaux Kir4.1. En outre, ces modifications sont associées une augmentation de la concentration extracellulaire de K+, qui in vitro,est responsable d'une augmentation de l'excitabilité neuronale. Par ailleurs, la déplétion virale des canaux Kir4.1 dans les astrocytes trigéminaux mime les effets du CFA en déclenchant une allodynie mécanique persistante et une augmentation de l’excitabilité neuronale, tandis que la restauration des canaux Kir4.1 sur des animaux CFA prévient l’allodynie mécanique et l’hyperexcitabilité des neurones trigéminaux. L’ensemble des résultats montre pour la première fois que le déficit du canal astroglial Kir4.1 dans le Sp5C sous-tend l'allodynie mécanique inflammatoire, et pourrait donc offrir de nouvelles pistes thérapeutiques pour ce symptôme invalidant<br>Chronic inflammatory pain is a frequent and disabling condition that is significantly maintained by centralsensitization, which results in pain hypersensitivity. While microgliosis seems to contribute to the onset ofchronic pain, reactive astrocytes and associated chemical mediators play a critical role in chronic painmaintenance and central sensitization.Inward rectifier potassium channels Kir4.1 are mainly expressed by astrocytes and are known to beinvolved in astrocytic physiological functions, particularly by controlling their resting membrane potential,but also in astrocyte-neuron interactions by controlling the glutamate uptake and extracellular potassium(K+) concentration. Accordingly, it has been shown that dysfunction of these channels is associated withseveral central nervous system pathologies including epilepsy, Huntington disease, depression or lateralamyotrophic sclerosis. However, the role of central astroglial Kir4.1 in pain has not been investigated.Here, we explored the involvement of the medullary dorsal horn (MDH) astroglial Kir4.1 channels inthe maintenance phase of trigeminal inflammatory pain hypersensitivity. By combining biochemical,electrophysiological and behavioral techniques, we showed that MDH astrocytes from inflamed rats havesmaller Kir4.1 currents, correlated with a decrease in Kir4.1 protein expression. Moreover, thesemodifications are associated with elevated extracellular K+ concentration, which increased MDH neuronalexcitability in vitro. Viral delivery of a dominant negative form of Kir4.1 channels to MDH astrocytesmimics CFA effects by inducing neuronal hyperexcitability and mechanical allodynia. Finally, restorationof Kir4.1 function recovered aspects of neuronal dysfunction and attenuated pain behaviors in CFA-injectedanimals.Together, these findings suggest that astroglial Kir4.1 channel deficit underlies inflammatory staticmechanical allodynia, and thus may offer novel avenues for the treatment of this debilitating symptom
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Xie, Lai-Hua. "Wortmannin, an inhibitor of phosphatidylinositol kinase, blocks the MgATP-dependent recovery of Kir6.2-SUR2A channel." Kyoto University, 1999. http://hdl.handle.net/2433/181696.
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Chever, Oana. "Implication du canal glial Kir4.1 dans la régulation du potassium extracellulaire : étude in vivo chez la souris knock-out Kir4.1 sous anesthésie." Thesis, Université Laval, 2008. http://www.theses.ulaval.ca/2008/25879/25879.pdf.
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Principalli, Maria Antonietta. "Etude structure-fonction du canal Kir6.2 et de son couplage avec des partenaires naturels et artificiels." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV014/document.
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Les canaux potassiques sensibles à l'ATP (K-ATP) jouent un rôle fondamental au sein de la cellule, puisqu'ils ajustent le potentiel de membrane en fonction de l'état métabolique. Ils combinent deux types de protéines: le récepteur des Sulfonylurée (SUR), protéine régulatrice faisant partie des transporteurs ABC, et le canal potassique rectifiant entrant Kir6. Elles s'associent en formant un hétérooctamère (4 SUR/4 Kir6) d'une taille de ~ 1MDa. A l'heure actuelle, l'unique structure disponible de ce complexe est une structure basse-résolution de 18 Å qui ne permet pas de visualiser correctement l'arrangement des différentes sous-unités. Le but principal de ce projet de thèse était d'obtenir des informations à la fois structurales et fonctionnelles sur le couplage entre Kir6.2 et SUR.Il existe 2 isoformes du Kir6 humain (Kir6.1 et 6.2) et 3 isoformes de SUR : SUR1, principalement exprimée avec Kir6.2 dans les cellules β pancréatiques et les neurones ; SUR2A, très abondante avec Kir6.1 dans les muscles cardiaques et squelettiques ; et SUR2B, présent avec Kir6.1 au niveau des muscles lisses. La façon dont SUR est capable de moduler l'ouverture du canal en réponse à la fixation d'un ligand est encore mal comprise.Au sein du canal K-ATP, SUR a un rôle de modulateur du gating de Kir6.2. Il a été montré que trois résidus (E1305, I1910, L1313) dans SUR2A, étaient impliqués dans la « voie d'activation » liant la fixation d'un ligand sur SUR2A et l'ouverture du canal Kir6. Afin d'examiner le rôle des résidus correspondants au sein de SUR1, nous avons réalisé des chimères entre SUR1 et le transporteur ABC MRP1 (qui n'interagit pas avec Kir6.2) et utilisé la technique du patch-clamp pour évaluer leur fonctionnalité. Nos résultats ont montré que les mêmes résidus au sein de SUR1 et SUR2A sont impliqués dans l'association fonctionnelle avec Kir6.2, mais que les spécificités au niveau de la chaine latérale pourraient expliquer les propriétés propres aux canaux pancréatiques et cardiaques. En effet, dans le pancréas, les canaux SUR1/Kir6.2 sont partiellement actifs au repos tandis que les canaux SUR2A/Kir6.2 du cœur sont principalement fermés. Cette spécificité peut être expliquée par les interactions spécifiques de SUR1 et SUR2A avec Kir6.2.La participation du canal Kir6.2 dans le couplage avec SUR ne peut être facilement étudiée puisque la région allant du N-terminal de Kir6.2 jusqu'à sa première hélice est physiquement associée à SUR. Des mutations à ce niveau pourraient affecter à la fois l'interaction physique et fonctionnelle avec SUR. Pour passer outre cet obstacle, nous avons utilisé la technologie ICCR développée dans notre laboratoire. Les ICCRs sont des protéines artificielles créées par couplage physique du C-terminal d'un RCPG au N-terminal de Kir6.2. Cette technologie permet l'étude de la fonction du N-ter de Kir6.2 puisque la fusion entre le RCPG et le canal assure une association fonctionnelle : le signal électrique généré par le canal ionique est directement lié à la fixation du ligand sur le RCPG. Le domaine reliant les deux protéines est essentiel pour la fonction de l'ICCR et sa longueur affecte la régulation du canal. De façon intéressante, deux ICCRs de même longueur mais ayant 9 résidus de différence présentent deux phénotypes différents : un fonctionnel, un inactif. L'ICCR inatif est caractérisé par la perte des résidus 26 à 34 du N-ter contenant 5 arginines. Nous avons réalisé la cartographie fonctionnelle de ces résidus essentiels pour la régulation de Kir6.2. Successivement, nous avons effectué les mêmes mutations d'arginines au sein du canal naturel K-ATP, mais n'avons pas observé de différence entre le canal muté et sauvage. Ces résultats suggèrent qu'il existe au moins deux voie de régulation pour le gating de Kir6.2 : une via les arginines du N-ter (utilisé par les RCPGs) et l'autre, toujours inconnue, utilisée par SUR<br>ATP-sensitive potassium (K-ATP) channels play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the SulfonylUrea Receptor (SUR), a protein of the ABC transporters family, and the inward rectifier K+ channel Kir6. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6) of ~ 1MDa. A high-resolution structure of the complex is still missing. To date, only a 18 Å structure of the full complex is available. Unfortunately, the low resolution prevent visualization of subunits arrangement. This PhD project aimed at obtaining structural and functional information on the functional coupling between Kir6.2 and SUR. Structural studies are still in progress.While 2 isoforms of the human Kir6 protein exists (Kir6.1 and 6.2), 3 isoforms of the SUR protein are known: SUR1, mostly expressed in pancreatic β-cells and neurons mainly with Kir6.2, SUR2A, abundant in cardiac and skeletal muscle mainly with Kir6.2, and SUR2B, found in smooth muscle mostly with Kir6.1. How SUR modulates channel gating in response to the binding of ligands is still poorly understood.The SUR protein belongs to a family of transporters but in K-ATP works as a gating modulator. How a 'transporter' modulate Kir6 gating? In SUR2A three residues (E1305, I1310, L1313) were found to be implicated in the ‘activation pathway' linking binding of openers to SUR2A and channel opening. To examine the role of the matching residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter MRP1 (which does not interact with Kir6.2), and used patch clamp to assess the functionality of SUR1/MRP1 K-ATP chimeric channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display side-chain specificities that could account for the contrasted properties of pancreatic and cardiac K-ATP channels. In fact, in pancreas, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2.The participation of the Kir6.2 channel in the coupling with SUR cannot be easily studied, as the region spanning from Kir6.2 N-terminal to its first helix is in thigh physical association with SUR. Mutations at this level could affect both physical and functional interaction with the regulatory subunit. To overcome this obstacle we used the ICCR technology developed in our laboratory. ICCRs are artificial proteins created by physical and functional linkage of a GPCR C-terminus to the Kir6.2 N-terminus. ICCRs provide a unique method to study the function of the Kir6.2 channel N-terminal, as the fusion between GPCR and channel ensure physical association. In ICCRs the electrical signal generated by the ion channel is directly linked to ligand binding on the GPCR. The domain linking GPCR and channel is crucial for ICCR function and its length affects channel regulation. Interestingly, two ICCRs, having identical linker length but nine residues differences at the fusion point, showed different phenotypes: one functional, one inactive (no channel regulation). The inactive ICCR is characterized by the lack of residues 26 to 34 in the channel N-terminus containing 5 arginines. We functionally mapped these arginines and identify specific residues essential for Kir6.2 regulation. Successively, we transferred this knowledge to the K-ATP mutating the previously found essential arginines. Here, we did not observe any change compared to wild-type channels. This result suggest that there are at least two ways to modulate Kir6.2 gating: one through the arginines in the N-terminal (used by the GPCR) and another, still unknown, used by SUR
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Tu, Wei [Verfasser], and Jürgen [Akademischer Betreuer] Daut. "Endozytose des Kaliumkanals Kir2.1 / Wei Tu. Betreuer: Jürgen Daut." Marburg : Philipps-Universität Marburg, 2015. http://d-nb.info/1076865720/34.
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Ju, Min. "Structure and function of Kir2.3, Kv2.1 and heag2 potassium channels." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405674.
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Ninomiya, Tomonori. "Verapamil, a Ca[2+] entry blocker, targets the pore-forming subunit of cardiac type K[ATP] channel (Kir6.2)." Kyoto University, 2004. http://hdl.handle.net/2433/147501.
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Agasid, Mark Tadashi, and Mark Tadashi Agasid. "I. Development of Rapid Conductance-Based Protocols for Measuring Ion Channel Activity; II. Expression, Characterization, and Purification of the ATP-Sensitive, Inwardly-Rectifying K+ Channel, Kir6.2, and Ion Channel-Coupled Receptors." Diss., The University of Arizona, 2017. http://hdl.handle.net/10150/623173.
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Ligand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).
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Papanikolaou, Maria. "Expression and function of Kir7.1 in the murine central nervous system." Thesis, University of Portsmouth, 2014. https://researchportal.port.ac.uk/portal/en/theses/expression-and-function-of-kir71-in-the-murine-central-nervous-system(5d95b958-7bcc-4ae5-8f8b-3f90a34d1c3f).html.
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Glia express a variety of ion channels, but the precise subtypes expressed by astrocytes and oligodendrocytes has not been fully elucidated. The Kir7.1 subtype of inwardly rectifying potassium channels (K<sub>ir</sub>) is highly expressed in retinal pigment epithelium and has been demonstrated in Purkinje neurons of the adult rat cerebellum and pyramidal neurons of the hippocampus, but it has not previously been identified in glia. Using quantitative real time PCR, an ion channel profile for the developing mouse optic nerve was constructed and K<sub>ir</sub>7.1 was identified as one of the major ion channels present. Immunostaining revealed widespread expression of K<sub>ir</sub>7.1 in glia and neurons in the mouse brain with the highest expression found in optic nerve oligodendrocytes. A major function of K<sub>ir</sub> is to maintain the membrane potential of glia in the face of large ionic shifts associated with normal neuronal function and pathology. Oligodendrocytes are particularly susceptible to ischemia so the role of Kir7.1 in maintaining oligodendrocyte integrity during oxygen and glucose deprivation (ODG) in the isolated intact mouse optic nerve was examined, using the K<sub>ir</sub>7.1 channel blocker VU590. Blockade of K<sub>ir</sub>7.1 resulted in increased cell death of optic nerve oligodendrocytes in normoxic conditions by activating caspase -dependent apoptotic pathways and significantly augmented cell death induced by OGD. Moreover, intracellular calcium fluctuations dependent on store operated calcium entry in optic nerve glia were identified as a potential mechanism for the cellular stress induced by K<sub>ir</sub>7.1 inhibition. The results presented within this thesis demonstrate functional expression of K<sub>ir</sub>7.1 in glial cells, and indicate they are important in maintaining oligodendrocytic integrity in both physiological and pathological conditions.
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Grothus, Katrin [Verfasser], and Jürgen [Akademischer Betreuer] Daut. "The serine/threonine protein kinase SGK3 stimulates endosomal recycling of the potassium channel Kir2.2. / Katrin Grothus. Betreuer: Jürgen Daut." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/109959426X/34.
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Stonehouse, Anthony Harold. "Expression of the inwardly rectifying potassium ion channel, Kir2.2, and its modulating receptor, NK-1 R, in rat brain." Thesis, University of Leicester, 1999. http://hdl.handle.net/2381/29639.
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Potassium current flowing through channels of the Kir2.0 subfamily of inward rectifier channels contributes to resting membrane potential in many cell types. Suppression of inward rectifier (Kir) activity by neurokinin neurotransmitters increases neuronal excitability. Despite functional studies reporting modulation of whole-cell Kir current in the locus coeruleus (LC) of the medulla, little information is available describing the specific molecules involved. Isoform specific polyclonal antibodies were raised, characterised and utilised, revealing differential distribution patterns. (a) Kir2.2 protein was observed in the cerebellum and medulla: a similar distribution was obtained by in situ hybridisation with Kir2.2 riboprobes. (b) Kir2.1, Kir2.2 and Kir2.3 were expressed in the hippocampus. Detailed observations of tissue sections revealed immunostaining patterns in macroglia, endothelia, ependyma and vascular smooth muscle cells for Kir2.1 and Kir2.2. The NK-1 receptor was localised to plasma membranes of neurons, neuronal processes and oligodendroglia. Kir2.0 channels appeared to be distributed on plasma membranes and within the nucleus. This intracellular distribution was mimicked in transiently transfected Chinese hamster ovary cells expressing Kir2.0 isoforms. Channel and receptor co-localisation supported the hypothesis that neuronal excitability in locus coeruleus is controlled by substance P-initiated suppression of Kir2.0 channel activity via protein phosphorylation. Tyrosine phosphorylation of Kir2.2 was investigated after administration of SP, in SN56 cell cultures. This produced a 62 kDa band, detectable by anti-Kir2.2 and anti-phosphotyrosine antibodies indicating that Kir2.2 protein could be phosphorylated via events initiated by SP.
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Dourado, Renata Montoro. "A diversidade alélica do gene KIR3DL2 e o seu impacto nos níveis de expressão gênica deferencial." reponame:Repositório Institucional da UFPR, 2017. http://hdl.handle.net/1884/47595.
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Orientadora : Profª. Drª. Karin Braun Prado<br>Coorientador : Prof. Dr. Danillo G. Augusto<br>Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 28/03/2017<br>Inclui referências : f. 92-102<br>Resumo: A familia de genes KIR (do ingles, killer cell immunoglobulin-like receptors) desempenha um papel central na imunidade inata e adaptativa e seu polimorfismo tem sido associado a suscetibilidade diferencial a diversas doencas. Esses genes exibem extensa variabilidade, tanto em termos de ausencia e presenca de genes, quanto em nivel alelico. Os receptores codificados por esses genes sao expressos na superficie das celulas NK e de alguns subtipos de celulas T e podem transduzir sinais ativadores ou inibidores. Pouco se conhece a respeito dos seus niveis de expressao genica diferencial, tampouco dos mecanismos de regulacao. O gene KIR3DL2 codifica um receptor inibidor e e o segundo gene KIR mais polimorfico e polialelico do complexo, com mais de 80 alelos descritos. O objetivo desse trabalho foi analisar se o polimorfismo alelico de KIR3DL2 impacta na sua expressao genica diferencial. Para isso, caracterizamos a diversidade alelica de KIR3DL2 em uma populacao de euro-descendentes de Curitiba e regiao metropolitana (n = 235), atraves de sequenciamento de DNA. As frequencias genotipicas estavam de acordo com as esperadas pelo equilibrio de Hardy-Weinberg, sendo os genotipos mais comuns: 3DL2*001/007 (11,5%), 3DL2*001/002 (6,8%) e 3DL2*002/007 (6,8%). Os alelos mais frequentes encontrados nessa populacao foram 3DL2*007 (21,7%), 3DL2*001 (21,3%) e 3DL2*002 (20%). Um total de 40 individuos com genotipos especificos e comuns na populacao desse estudo foram selecionados para a analise de expressao diferencial por citometria de fluxo. A analise de expressao diferencial foi realizada com os individuos portadores dos alelos mais frequentes encontrados na populacao, sendo eles: 3DL2*001, 3DL2*002, 3DL2*003, 3DL2*005, 3DL2*007, 3DL2*009, 3DL2*010 e 3DL2*011. Verificamos que o polimorfismo alelico de KIR3DL2 esta associado nao somente com niveis de expressao diferencial, mas tambem com diferentes quantidades de celulas NK que exibem KIR3DL2 em sua superficie. O alelo KIR3DL2*002 foi associado ao maior nivel de expressao de KIR3DL2 e ao maior numero de celulas NK 3DL2 positivas. Ja 3DL2*010 foi associado ao menor nivel de expressao e a menor quantidade de celulas NK com KIR3DL2 presente na superficie celular. Alem disso, demonstramos que um polimorfismo na regiao 3' UTR, na posicao 16545A>G (rs1865095) marca os niveis de expressao de KIR3DL2. Sugerimos que esta expressao diferencial possa estar relacionada a ligacao de miRNAs nesta regiao, especificamente o miR-2114-3p. A presenca de ligantes especificos (HLA-A3, -A11 e -B27) nao esta associada a diferentes niveis de expressao de KIR3DL2 (p = 0,5739) nem a diferentes quantidades de celulas NK KIR3DL2 positivas (p = 0,7772). Alem disso, nenhuma correlacao foi encontrada entre a expressao diferencial dos alelos de KIR3DL2 e a expressao diferencial dos alelos de HLA-A. Como perspectivas futuras pretendemos analisar, atraves de co-cultivo celular, a atividade citotoxica das celulas NK de individuos com diferentes genotipos homozigotos, como KIR3DL2*001/KIR3DL2*001, KIR3DL2*002/KIR3DL2*002, KIR3DL2*007/KIR3DL2*007 e KIR3DL2*010/KIR3DL2*010 a fim de corroborar as hipoteses geradas. Palavras-chave: Celulas NK, KIR3DL2, variabilidade alelica, HLA-A3, HLA-A11, HLA-B27, expressao diferencial.<br>Abstract: The KIR (killer cell immunoglobulin-like receptors) gene family plays a central role in innate and adaptive immunity and has been associated with differential susceptibility to diseases. Besides the uncommon presence and absence polymorphism that occurs in KIR, these genes also exhibit an extensive allelic variation. The receptors encoded by these genes are expressed on the surface of NK cells and on some subset of T cells. They can transduce either activating or inhibiting signals. The differential expression levels and the mechanisms of genetic regulation of these receptors are poorly known. The KIR3DL2 gene encodes an inhibitory receptor and it is one of the most polymorphic and polyallelic KIR, with more than 80 alleles described so far. This study aimed to analyze if there are a profound impact of KIR3DL2 allelic polymorphism on its differential gene expression. Allelic diversity of KIR3DL2 was characterized in an euro-descendant population from Curitiba and metropolitan region, state of PR (n = 235), by sequencing-based typing. Genotype frequencies were in accordance with Hardy-Weinberg equilibrium and the most frequent genotype was 3DL2*001/007 (11.5%), followed by 3DL2*001/002 (6.8%) and 3DL2*002/007 (6.8%). The most frequent alleles in the population were 3DL2*007 (21.7%), 3DL2*001 (21.3%) and 3DL2*002 (20%). A total of 40 individuals with specific and commom genotypes were selected for differential expression analysis by flow citometry. The differential expression analysis was made for the most common alleles found in the population: 3DL2*001, 3DL2*002, 3DL2*003, 3DL2*005, 3DL2*007, 3DL2*009, 3DL2*010 and 3DL2*011. We verified that the allelic polymorphism of KIR3DL2 was associated not only with the differential expression but also with the different amount of NK cells that display KIR3DL2 on their surface. KIR3DL2*002 allele was associated with higher expression of KIR3DL2 and higher number of KIR3DL2 positive NK cells, while KIR3DL2*010 was related to the lowest expression and lowest level of KIR3DL2 positive NK cells. It has also been found that a polymorphism in the 3' UTR, 16545A>G (rs1865095), was associated with KIR3DL2 differential expression level. We suggest that it may be related to miRNAs binding, specifically miR-2114-3p. The presence of specific HLA class I ligands (HLA-A3, -A11 and -B27) was not associated with KIR3DL2 differential expression levels (p = 0.5739) nor with different amount of NK KIR3DL2+ cells (p = 0.7772). In addition, no correlation was found between the differential expression of the KIR3DL2 alleles and the differential expression of the HLA-A alleles. As future perspectives, it is intended to analyze the citotoxic activity of NK cells from individuals with different genotypes like KIR3DL2*001/KIR3DL2*001, KIR3DL2*002/KIR3DL2*002, KIR3DL2*007/KIR3DL2*007 and KIR3DL2*010/KIR3DL2*010 through co-culture to corroborate the hypotheses generated. Key-words: NK cells, KIR3DL2, allelic variability, HLA-A3, HLA-A11, HLA-B27, differential expression.
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Chen, Shen. "Ventricular myocyte sarcolemmal Kir6.1/SUR2B potassium channels : a potential effector of cardioprotection." Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42498.
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KATP channels are ubiquitously expressed and are said to couple cellular metabolic state and electrical excitability. The KATP channel complex is composed of two subunits in a hetero-octameric structure, with Kir6 subunit form the pore and sulphonylurea receptors (SUR) as β-subunits. In the heart, KATP channels are considered to be composed of the Kir6.2/SUR2A combination and play a key role in responding to metabolic stress as well as in cardioprotection. It is hypothesised that opening of the cardiac SarcoKATP channel shortens the action potential duration so reducing the ATP consumption during an ischaemic event to preserve energy and so limit Ca2+ overload. Previous data (unpublished) suggested there was a Kir6.1-like current, with constitutive activity, on the rat cardiomyocyte sarcolemmal membrane. In this study, firstly, the Kir6.1-like channel was formally identified by using electrophysiology patch clamp and specific pharmacological inhibitors. Secondly, further evidence was acquired using specific knockdown the pore-forming and β-subunits using adenoviral-introduced shRNA. Finally, for SarcoKir6.1 current was investigated in control cardiomyocytes and in IPC, adenosine-induced cardioprotection and sex-specific cardioprotection. In this study, a new small-conductance KATP channel was identified at the cardiac sarcolemmal surface along with the large-conductance channel. These findings suggest that there are two components to IKATP in the heart, IKATP-SC, composed of Kir6.1/SUR2B subunits, and IKATP-LC which is the classically thought of cardiac Kir6.2/SURA complex. It is suggested the newly discovered IKATP-SC plays an important role in regulating action potential duration as well as mediating cardioprotection. This IKATP-SC may help explain the role that cardiac sarcolemmal KATP plays in cardioprotection.
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Rodzli, Nazahiyah. "Structural and interaction studies of PSD95 PDZ domain-mediated Kir2.1 clustering mechanisms." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/structural-and-interaction-studies-of-psd95-pdz-domainmediated-kir21-clustering-mechanisms(b07686a7-7a91-4673-bed3-16707f502c61).html.
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PSD95 is the canonical member of the Membrane Associated Guanylate Kinase class of scaffold proteins. PSD95 is a five-domain major scaffolding protein abundant in the postsynaptic density (PSD) of the neuronal excitatory synapse. Within PSD95 three PDZ domains modulate protein-protein interactions by selectively binding to short peptide motifs of target proteins. Under the direction of the multivalent PDZ domain interactions, the interacting proteins tend to cluster at the PSD, a phenomenon that is critical for synaptic signalling regulation. Earlier studies have shown that the N-terminal PDZ domains of PSD95 are obligatory for the clustering to occur. This thesis focuses on the strong inwardly rectifying potassium channel, Kir2.1 as the PSD95 binding partner. Kir2.1 is known to maintain membrane resting potential and control cell excitability. Previous studies have reported that Kir2.1 clustered into ordered tetrad complexes upon association with PSD95.This study investigates the detailed clustering mechanisms of Kir2.1 by PDZ domains. To achieve this, components that are involved in the formation of a complex namely PSD95 sub-domains comprising single PDZ and the tandem N terminal PDZ double domain (PDZ1-2), and Kir2.1 cytoplasmic domains(Kir2.1NC) are studied in detail via different structural and biophysical approaches; 1) PDZ1-2 is examined in apo- and bound ligand form with a Kir2.1 Cterminal peptide in crystal and solution via X-ray crystallography and small angle X-ray scattering; 2) the tandem and the single PDZ domain interaction with ligand are measured thermodynamically via isothermal calorimetry (ITC); 3) the complex of full length PSD95 with Kir2.1NC is analyzed with electron microscopy (EM). The protein components are produced in high quality by protein expression and multiple-step protein purification techniques. PDZ1-2 crystallographic structures were solved at 2.02A and 2.19A in theapo- and the liganded forms respectively. The solution state analysis showed domain separation and structural extension of the tandem domain when incorporated with the ligand. The ITC experiment revealed PDZ1-2 to have greater affinity towards the peptide ligand relative to the single PDZ domains. These combinatorial outcomes lead to the conclusion that PSD95 clusters Kir2.1 by adopting an enhanced binding interaction which is associated with increased PDZ1-2 inter-domain separation. The preliminary analysis of PSD95-Kir2.1NC complex with cryo-EM showed the establishment of a tetrad and led to a reconstruction at 40A resolution. The work in obtaining a higher resolution complex structure is promising with further data collection required to allow the employment of more sophisticated model reconstruction processes.
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Kono, Yutaka. "The propeties of the Kir6.1-6.2 tandem channel co-expressed with SUR2A." Kyoto University, 2001. http://hdl.handle.net/2433/150525.
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Pini, Jonathan. "Modélisation du syndrome d'Andersen dans les cellules souches pluripotentes induites : implication du canal potassique Kir2.1 dans la morphogenèse osseuse." Thesis, Nice, 2016. http://www.theses.fr/2016NICE4042/document.
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Le syndrome d’Andersen est une maladie rare et associée à la perte de fonction du canal potassique Kir2.1. Afin d’étudier sa physiopathologie, nous avons généré et caractérisé des cellules souches pluripotentes induites (iPS) contrôle et Andersen. Nous avons ensuite différencié ces cellules iPS en cellules souches mésenchymateuse (MSC). Les cellules MSC de patients présentent une capacité de différenciation en ostéoblastes et en chondrocytes diminuée par rapport aux cellules contrôle. En effet, la production de matrice extracellulaire et l'expression des master gènes des différenciations osseuses et cartilagineuses, est réduite chez les patients. Ces travaux de thèse montrent que le canal Kir2.1 est essentiel au développement osseux. Les défauts de différentiation observés pourraient expliquer les dysmorphies associées avec le syndrome d’Andersen<br>Andersen's syndrome is a rare disorder associated with a Kir2.1 potassium channel loss of fuction. To study the pathophysiology, we have generated and characterized induced Pluripotent Stem cells (iPS) from control and patient cells. We have then differentiated those iPS cells into mesenchymal stem cells (MSC). Patient's MSc have a lower osteoblastic and chondrogenic differnciation ability compared to control cells. Indeed, extracellular matrix production and master gene expression of osteoblastic and chondrogenic differenciation are reduced in patient’s cells. Alltogether, these results shown that Kir2.1 channel is required for bone developement. The differenciation defects saw in patient cells could explain the Andersen's syndrome associated dysmorphies
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Matonti, Julia. "Rôle du canal potassique Kir2.1 dans la morphogenèse osseuse : Modélisation du syndrome d'Andersen." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://www.theses.fr/2019AZUR6009.
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Le syndrome d'Andersen (AS) est un trouble complexe caractérisé par une triade de symptômes: des paralysies périodiques, des arythmies cardiaques, et des troubles du développement (Andersen 1971). C’est une pathologie rare, héréditaire dominante, associée à des mutations génétiques du gène KCNJ2 (Plaster 2001) qui encode le canal potassique à rectification entrante Kir2.1. Le Kir2.1 est connu pour jouer un rôle majeur dans la stabilisation du potentiel de repos et dans la phase tardive de repolarisation du potentiel d’action cardiaque. Les caractérisations fonctionnelles de ces mutations ont été réalisées in vitro et ex vivo et ont montré l’effet dominant négatif des mutations sur ce canal. Des progrès ont été faits pour comprendre les rôles de ce canal dans les tissus musculaires et cardiaques (Zaritsky 2001). Cependant, peu d’études ont abordé le rôle du canal Kir2.1 dans les manifestations développementales et notamment son rôle dans les cellules non-excitables. De plus, l’invalidation (KO) du gène KCNJ2 chez la souris est létale quelques heures après sa naissance, due à une fente palatine (Zaritsky 2000, Dahal 2012). Cette mortalité post-natale précoce rend les études in vivo difficiles, notamment celles visant à évaluer son rôle dans les tissus adultes. Une alternative est l’utilisation d’un modèle in vitro impliquant des cellules pluripotentes induites humaines (iPS) (Takahashi 2007) qui sont capables d’être différenciées dans tous les types cellulaires. L’équipe du Dr. Bendahhou a précédemment montré que le canal fonctionnel Kir2.1 était nécessaire à la formation de l’os, dans un modèle de différentiation de myoblastes en ostéoblastes (Sacco 2015). De plus, ils ont généré des iPS à partir de biopsies musculaires de personnes saines et de patients atteints du AS (Pini 2016). Nous avons également montré que l’absence du Kir2.1 dans la différenciation ostéoblastique et chondrocytaire impactait la production de matrice mais aussi l’expression des gènes principaux de ces deux lignages (Pini 2018). Dans l’ostéoblastogenèse, l’activité́ de la voie de signalisation Bone Morphogenetic Protein (BMP) est diminuée dans les cellules AS (Pini 2018). Mon projet se focalise sur la détermination du rôle du canal Kir2.1 dans la voie de signalisation BMP dans les ostéoblastes in vitro. Pour ce faire, j’ai utilisé deux types cellulaires. En profitant de leur capacité de différenciation, j’ai utilisé les iPS (saines et AS) déjà générées par l’équipe, mais aussi des iPS générées par mes soins, afin d’étudier le rôle du Kir2.1 dans les tissus osseux. En parallèle, j’ai utilisé une lignée d’ostéoblastes humains fœtaux immortalisés (hFOB) mis au point par Harris en 1995. Une combinaison d'analyses transcriptomiques et protéomiques, ainsi que d’immunohistochimie, ont été utilisées pour déterminer le rôle du canal potassique Kir2.1 dans les étapes clés de la morphogenèse osseuse<br>Andersen's syndrome (AS) is a complex disorder characterized by a triad of symptoms: periodic paralysis, cardiac arrhythmias, and developmental disorders (Andersen 1971). It is a rare pathology, dominant hereditary, associated with genetic mutations of the KCNJ2 gene (Plaster 2001) that encodes the inward rectifier potassium channel Kir2.1. Kir2.1 is known to play a major role in stabilizing resting cell potential and in the late phase of repolarization of cardiac action potential. Functional characterizations of these mutations were performed in vitro and ex vivo and showed the dominant negative effect of mutations on this channel. Progress has been made in understanding the roles of this channel in muscle and heart tissue (Zaritsky 2001). However, few studies have addressed the role of the Kir2.1 channel in developmental manifestations, including its role in non-excitable cells. In addition, the invalidation (KO) of the KCNJ2 gene in mice is lethal within hours of birth due to cleft palate (Zaritsky 2000, Dahal 2012). This early post-natal mortality makes in vivo studies difficult, particularly those to assess its role in adult tissues. An alternative is the use of an in vitro model involving human induced pluripotent cells (iPS) (Takahashi 2007) that are capable of being differentiated in all cell types. Dr. Bendahhou's team has previously shown that the Kir2.1 functional channel is necessary for bone formation in a model of myoblast differentiation into osteoblasts (Sacco 2015). In addition, they generated iPS from muscle biopsies of healthy individuals and AS patients (Pini 2016). We have also shown that the absence of Kir2.1 in osteoblastic and chondrocytic differentiation impacts not only matrix production but also the expression of the main genes of these two lineages (Pini 2018). In osteoblastogenesis, activity of the Bone Morphogenetic Protein (BMP) signaling pathway is decreased in AS cells (Pini 2018). My project focuses on determining the role of the Kir2.1 channel in the BMP signalling pathway in in vitro osteoblasts. To do this, I have used two cell types. Taking advantage of their differentiation capacity, I used the iPS (healthy and AS) already generated by my team, but also the iPS I have generated, to study the role of Kir2.1 in bone tissue. In parallel, I used a cell line of immortalized fetal human osteoblasts (hFOB) developed by Harris in 1995. A combination of transcriptomic and proteomic analyses, as well as immunohistochemistry, were used to investigate the role of the Kir2.1 potassium channel in key stages of bone morphogenesis
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Sako, Nouhoum. "Lymphome NK/T et lymphomes T cutanés : Recherche et analyse de marqueurs fonctionnels (HACE1, CD 160 et KIR3DL2) : Implication de marqueurs biologiques dans la physiopathologie." Paris 7, 2014. http://www.theses.fr/2014PA077231.
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Les lymphomes T sont des maladies souvent agressives, de mauvais pronostic et résistantes aux traitements actuels. Dans la première partie de ce travail, j'ai étudié l'implication possible dans la physiopathologie du lymphome NK/T extra ganglionnaire de type nasal d'un gène suppresseur de tumeur potentiel (HACE1) associé à la délétion fréquente observée en 6q21. Les études ont montré que HACE1 (une E3 ubiquitine ligase), impliquée dans la régulation de plusieurs protéines importantes dans la biologie cellulaire, est sous exprimée au niveau transcrit (ARNm) chez ces malades et les lignées cellulaires. Par un faisceau d'arguments, j'ai montré que HACE1, bien qu'il soit sous exprimé au niveau ARNm, présente une protéine en quantité suffisante et parfaitement fonctionnelle. J'ai également montré que la surexpression de la protéine HACE1 dans les lignées de lymphome T n'a pas de conséquences ni sur la survie, ni la prolifération cellulaire. J'ai réorienté notre projet de recherche, à la suite de ces résultats, en défaveur de HACE1, sur les études de certains marqueurs NK (CD160, KIRDL2) retrouvés sur les cellules tumorales des lymphomes T cutanés. Le KIR3DL2 et le CD160 sont des marqueurs récepteurs CMH-I respectivement inhibiteur et activateur des cellules NK. Dans cette deuxième partie, j'ai identifié une population de cellules T dans la peau normale qui exprime ces deux marqueurs et pourrait être l'origine de la cellule tumorale dans le lymphome cutané. Mes résultats m'ont également permis d'émettre l'hypothèse de l'évolution de l'expression de ces 2 marqueurs avec le CD160 un marqueur des stades précoces et le KIR3DL2 un marqueur des stades tardifs du mycosis fongoïde<br>T-cell lymphomas are aggressive diseases with resistance to current treatments and poor prognosis. In the first part of this work, I studied the possible involvement of tumor suppressor gene (HACE1) associated with the common deletion observed in 6q21 in the pathogenesis of NK / T extra nodal nasal type lymphoma (NKTCL). Research has shown that HACE1 (an E3 ubiquitin ligase), involved in the regulation of many important cellular proteins, is down expressed at the transcript level (mRNA) in these patients and cell Unes. By a range of argument, I showed that the endogenous protein of HACE1 is in fact present in sufficient quantities and functional. I also showed that overexpression of HACE1 protein in NKTCL cells Unes has no effect on either survival or cell proliferation. I refocused my research project on the study of some NK markers (CD160, KIR3DL2) found on cutaneous T cell lymphomas. The KIR3DL2 and CD160 are respectively MCH I inhibitor or activator receptor of NK cell. In this second part, I have identified a population of T cells CD4+ CD160+ KIR3DL2+ in normal skin, which could be the origin of cutaneous lymphoma. My results demonstrate that CD160 and KIR3DL2 are two important markers for cutaneous lymphocytes
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Djukic, Biljana McCarthy Ken D. "Role of the inward rectifying potassium channel, Kir4.1, in astrocyte physiology and neuronal excitability." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,270.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.<br>Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology." Discipline: Pharmacology; Department/School: Medicine.
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Konduru, Anuhya S. "The Reactive Carbonyl Methylglyoxal Suppresses Vascular KATP Channels by MRNA Destabilization." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_theses/34.
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Diabetes mellitus is characterized by hyperglycemia, oxidative stress and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl. MGO can readily interact with proteins, lipids and DNA, and cause an imbalance of the cellular antioxidant system leading to carbonyl stress. The effects of MGO can be devastating if the targeted molecules are responsible for the maintenance of membrane potentials and ionic homeostasis. Here we show that MGO disrupts the vascular isoform of ATP-sensitive K+ (KATP) channels by acting on the mRNAs of Kir6.1 and SUR2B subunits thereby regulating vascular tone. Our results show that the 3’ untranslated region (UTR) of Kir6.1 mRNA and the coding region of SUR2B mRNA are targeted by MGO causing a disruption of vascular KATP channels. The destabilization of the mRNAs of KATP channel can in turn affect K+ homeostasis of vascular smooth muscles as well as vascular responses to circulating vasodilators and vasoconstrictors.
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Wang, Kemeng. "Role of potassium channel Kir4.1(KCNJ10) in the wound healing of human corneal epithelial cells." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12245.
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Thesis (M.A.)--Boston University<br>The cornea, which is key to maintaining our normal vision by refracting light onto the lens and retina, as well as serving as a physical barrier to protect our eyes from the environment, contains epithelium tissue with one of the highest capacities for renewal and wound healing in the body. Although many studies have looked at the process of wound healing in corneal epithelium, most of them have focused on the various ligand-receptor growth factor signaling pathways, and few studies have been done to study the signaling and regulations of wound healing that are initiated intracellularly, or those associated with electrical currents and channel activity. This study, therefore, aims to look at the hypotheses that injury to the corneal epithelium leads to the downregulation of a type of potassium transport channels named Kir4.1/KCNJ10, and that the inhibition of KCNJ10 is associated with intracellular microRNA-205 (miR-205), which is upregulated during injury and healing. Together, the modulations in the level of KCNJ10 and microRNA-205 contribute to change in the environment
around the wound and promote the cellular processes that allow for efficient corneal healing.
To investigate the role of potassium channels and microRNAs in the cornea epithelium, an in-vitro model of endogenous wound healing was employed with human corneal epithelium cells (HCECs) serving as the primary model of study. Physiological injury was simulated using a scratch-wound model. The protein expressions for KCNJ10 and microRNA-205 were measured through various time points from both control and injured HCECs. The effect of two RNAi modulators of microRNA-205, a mimic and antagonist, and of KCNJ10 blockers were also tested for effects on the rate and efficiency of HCEC wound healing.
Results indicated that the expression of miR-205 increased in scratch-injured HCECs and that the expression of KCNJ10 decreased in wounded and healing HCECs. It was also shown that increasing KCNJ10 and decreasing miR-205 both lead to delayed healing, but that blocking KCNJ10 could partially abolished the effect of delayed healing associated with decreasing miR-205 and restored the healing process. It was also shown that the 3'UTR of KCNJ10 contains potential target sites for miR-205 binding and action. The results indicate that that KCNJ10 expression is negatively associated with corneal wound healing, and that miR-205 is upregulated upon injury in wounded corneal epithelium to inhibit KCNJ10 and allow for the processes of wound healing to take place.
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Aljohi, Mohammed. "Characterization of interaction sites between Kir6.0 and SUR subunits of ATP-sensitive potassium (KATP) channels." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29883.
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This study investigated cytoplasmic inter-subunit interactions between the Kir6.2 and SUR2A subunits of the cardiac ATP-sensitive potassium channel. The channels are a heterooligomeric complex of pore-forming Kir6.2 subunits and sulphonylurea receptor (SUR2A) subunits. Interactions between the cytoplasmic loops, the nucleotide binding domains (NBF1 and NBF2) of SUR2A and the full length of Kir6.2 were determined. In co-immunoprecipitation experiments, fragments from the C-terminal of SUR2A containing residues 1294-1358 tagged with Maltose-binding protein (MBP) showed binding with the full length Kir6.2 subunit, while residues between 1358-1545 did not. This indicated involvement of a 65 amino acid domain in the proximal C-terminal of SUR2A in forming a direct interaction with Kir6.2. When HEK 293 cells stably expressing Kir6.2/SUR2A channels were transiently transfected with SUR2A fragments containing residues 1294-1359, KATP current was decreased. This current reduction was due to a decreased number of channel subunits in the cell membrane; this was demonstrated by using immunocytochemistry, which showed that anti-K ATP channel subunit-associated fluorescence was lower in the cell membrane and increased in the intracellular compartment in the presence of the binding region.;Use of SUR2A/MRP1 chimaeras of the putative binding domain showed that the last eleven amino acids of the binding region were important for binding activity but that they do not contain all the elements necessary for binding. Co-immunoprecipitation and assays of disruption of functional channels with the binding domain chimaeras suggested an important role for the residues between 1318 and 1337 in the Kir6.2 binding motif within the SUR2A C-terminal domain.
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Zuniga, Dania. "Structural and functional studies of a human potassium channel, Kir2.1. Mechanism and consequences of mutations." Electronic Thesis or Diss., Sorbonne université, 2022. http://www.theses.fr/2022SORUS336.
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La capacité d'une cellule à faciliter le mouvement sélectif et rapide des ions et des petites molécules à travers la membrane plasmique est un processus biologique indispensable. Les canaux potassiques à rectification entrante (Kir) sont des protéines membranaires intégrales qui fournissent des voies sélectives aux ions K+ à travers la membrane cellulaire autrement imperméable, le long des gradients électrochimiques. Les canaux Kir permettent le passage des ions K+ dans les deux sens et régulent diverses fonctions chez les humains, notamment la fréquence cardiaque, le tonus vasculaire, la sécrétion d'insuline et l'équilibre salin et hydrique. L'importance physiologique des canaux Kir est mise en évidence par le fait que des défauts génétiquement héréditaires de ces gènes entraînent des maladies humaines (canalopathies). Nous nous intéressons particulièrement au syndrome d'Andersen, où des mutations du gène KCNJ2 codant pour la protéine Kir2.1 sont directement impliquées. Le syndrome d'Andersen (AS) est une maladie rare caractérisée par des arythmies cardiaques, des dysmorphies multiples et des paralysies périodiques. Le traitement disponible pour les patients atteints d’AS est empirique plutôt que rationnel en raison de la manque de connaissance de ce canal Kir2.1. Cette thèse visait à identifier les différences entre le canal Kir2.1 de type sauvage et deux canaux mutants causant l’AS afin de trouver des liens entre la structure et la fonction du Kir2.1 humain en utilisant une combinaison d'approches biochimiques, structurelles et fonctionnelles. Dans cette étude, nous avons exprimé le canal Kir2.1 humain dans la levure et l'avons purifié dans un détergent. Nous avons caractérisé l'interaction entre Kir2.1 et l'activateur essentiel PIP2, résolu par cryo-EM la première structure d'un canal Kir humain et exploré la reconstitution dans des systèmes modèles sans détergent comme les amphipols et les nanodisques. Les résultats de cette étude fourniront une base structurale et fonctionnelle pour mieux comprendre les mécanismes impliqués dans les canaux Kir et les effets de leurs mutations. Ce manuscrit est divisé en trois parties. La première partie présente les canaux Kir et l'état de l'art. La deuxième partie porte sur la caractérisation du canal potassique humain Kir2.1, la détermination de sa structure par cryo-EM et l'impact des mutations sur sa structure et sa fonction. La troisième partie présente deux mutations dans l'homologue bactérien KirBac3.1, qui partage des caractéristiques structurelles avec Kir2.1, pour donner un aperçu du mécanisme d’ouverture et fermeture des canaux Kir<br>The ability of a cell to facilitate the selective and rapid movement of ions and small molecules across the plasma membrane is one of the most fundamental biological processes. Inward rectifier potassium (Kir) channels are integral membrane proteins that provide K+-selective pathways across the otherwise impermeable cell membrane along the electrochemical gradients. Kir channels support the flow of K+ ions into and out of the cell and regulate various functions in the human body, including heart rate, vascular tone, insulin secretion, and salt and fluid balance. The physiological significance of Kir channels is highlighted by the fact that genetically-inherited defects in these genes result in human diseases (channelopathies). We are particularly interested in Andersen’s syndrome, where mutations in the KCNJ2 gene coding for Kir2.1 protein are directly involved. Andersen’s syndrome (AS) is a rare disease characterized by cardiac arrhythmias, dysmorphic features, and periodic paralysis. The available treatment for AS patients is empirical rather than rational due to the lack of knowledge of this Kir2.1 channel. This thesis aimed to identify the differences between the wild-type Kir2.1 channel and two mutant AS-causing channels to find links between the structure and the function of human Kir2.1 using a combination of biochemical, structural, and functional approaches. In this study, we recombinantly expressed the human Kir2.1 channel in yeast and purified it in detergent. We characterized the interaction between Kir2.1 and the essential activator PIP2, solved the first human Kir channel structure by cryo-EM, and explored reconstitution in detergent-free systems like amphipols and nanodiscs. The findings of this study will provide a structural and functional base to understand better the mechanisms involved in Kir channels and the effects of their mutations. This manuscript is divided into three parts. The first part introduces Kir channels and state-of-the-art. The second part focuses on the characterization of the human potassium channel Kir2.1, the determination of its structure by cryo-EM, and the impact of mutations on its structure and function. The third part presents two mutations in the bacterial homolog KirBac3.1, which shares structural features with Kir2.1, to provide insight into the gating mechanism of Kir channels
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Rojas, Asheebo. "KIR Channels in CO2 Central Chemoreception: Analysis with a Functional Genomics Approach." unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-08032007-163450/.
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Thesis (Ph. D.)--Georgia State University, 2007.<br>Title from file title page. Chun Jiang, committee chair; Delon Barfuss, Deborah Baro, Teryl Frey, committee members. Electronic text (226 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Nov. 1, 2007. Includes bibliographical references (p. 210-226).
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Stauß, Eva [Verfasser], and Ullrich [Akademischer Betreuer] Quast. "Die Mutation Y1206S steigert die Affinität des Sulfonylharnstoffrezeptors SUR2A für Glibenclamid und unterstützt den Einfluß der Koexpression mit KIR6.2 / Eva Stauß ; Betreuer: Ullrich Quast." Tübingen : Universitätsbibliothek Tübingen, 2006. http://d-nb.info/1160754330/34.
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Töpert, Christoph. "Struktur des KCNJ14-Gens und funktionelle Bedeutung der Kir2.4-Kanaluntereinheit beim Menschen und bei der Maus." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971231249.
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Abrams, Christopher John. "Studies of the molecular basis of selectivity and gating in the inward rectifier potassium channel Kir2.1." Thesis, University of Leicester, 2000. http://hdl.handle.net/2381/29921.
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1. The molecular basis of selectivity and gating were investigated in wild-type and mutant forms of the inward rectifier K+ channel Kir2.1 (IRK1). 2. Kir2.1 channels show characteristic time-dependent gating kinetics due to a reversible voltage-dependent channel block by cytoplasmic polyamines. Mutations at Asp 172 in the M2 domain revealed that a negative charge at this position is the main criterion of time-dependent gating kinetics in Kir2.1. 3. Kir channels are blocked by Cs+ and Rb+ in a voltage-dependent manner, characteristic of many Kir channels. Rb+ and Cs+ block in Kir2.1 was abolished by replacing Asp 172 by Asn, but was re-established by a change to Gln, narrowing the pore. However, blocking affinity was reduced by the mutation to Gln. 4. When Asp 172 was mutated to Glu, narrowing the pore but retaining the negative charge, block by both Cs+ and Rb+ was increased relative to wild-type. 5. Replacing Gly 168 in M2 by Ala was suggested to widen the pore at position 172. The effect of this mutation on Cs+ and Rb+ block was relatively small. 6. There appears to be a balance between charge and pore size in determining whether icons block or permeate. A major part of the selectivity of Kir2.1 is associated with Asp 172 in the M2 domain. This site also determines the time-dependent activation gating of the channel. 7. Mutation of Asp 172 to Ser were predicted to abolish both Cs+ and Rb+ block in Kir2.1, but blocking affinity was similar to WT. Therefore, other properties of the pore must contribute to Cs+ and Rb+ block at position 172 in addition to the electrostatic and steric effects identified in this study.
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Ghazi, Bouchra. "Réponses cellulaires associées au récepteur KIR3DL2, marqueur spécifique des lymphocytes T tumoraux du syndrome de Sézary." Thesis, Paris Est, 2012. http://www.theses.fr/2012PEST0068.
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Le syndrome de Sézary (SS) est un variant leucémique et érythrodermique de lymphomes T cutanés épidermotropes. Son diagnostic repose à la fois sur des critères cliniques, la présence de lymphocytes T à noyau atypique cérébriforme sur un frottis sanguin et la mise en évidence dans la peau, les ganglions et le sang d’un clone lymphocytaire T CD4+. Notre laboratoire a identifié KIR3DL2 comme premier marqueur membranaire spécifique des cellules tumorales de Sézary. KIR3DL2 peut ainsi être utilisé pour le diagnostic et le suivi des patients atteints du SS. Toutefois, aucune étude n’a démontré de lien entre sa structure de récepteur inhibiteur et sa fonction dans les lymphocytes tumoraux de Sézary, et plus particulièrement son implication possible dans les mécanismes régulant la prolifération et/ou la résistance à l’apoptose des cellules tumorales.Au cours de ce travail deux axes ont été développés :- Un premier axe visant à mieux comprendre la fonction de KIR3DL2 et les mécanismes de signalisation intracellulaire initiés lors de son engagement par l’anticorps AZ158 dans les lymphocytes T tumoraux de Sézary. Nos résultats mettent en évidence un rôle de corécepteur inhibiteur pour KIR3DL2 dans les cellules tumorales de Sézary. En effet, l’engagement de KIR3DL2 inhibe la prolifération et l’AICD induites par la stimulation CD3, cette inhibition étant corrélée à une modulation négative des signaux médiés par le TCR. Ainsi, KIR3DL2 ne se comporte pas comme une unité de signalisation indépendante dans les cellules tumorales de Sézary, contrairement à ce qui est observé dans les cellules NK.- Un second axe portant sur l’évaluation d’une nouvelle fonction de KIR3DL2 comme récepteur pour les ODN CpG. Ainsi, nous rapportons pour la première fois un effet direct de l’ODN CpG sur les cellules tumorales T CD4+ de Sézary. En effet, nous avons observé un effet apoptotique de l’ODN CpG-C caspases-dépendant sur les lignées et les cellules tumorales circulantes. De plus, le traitement des cellules tumorales de patients Sézary avec l’ODN CpG-C conduit à une inhibition de l’activation constitutive du facteur de transcription STAT3.La réalisation de cette étude a permis de mieux comprendre la fonction et les mécanismes initiés à partir de KIR3DL2 dans les cellules tumorales T CD4+ de Sézary. De plus, ce travail ouvre de nouvelles perspectives thérapeutiques basées sur le ciblage direct et spécifique des cellules tumorales de Sézary pouvant être associé à une stimulation des acteurs immuns grâce à l’action des ODN CpG<br>Sézary syndrome (SS) is an aggressive leukemic and erythrodermic variant of cutaneous T-cell lymphoma. It is characterized by the presence of a clonal CD4+ T lymphocyte population in the skin, lymph nodes and peripheral blood. Our laboratory has previously identified the NK cell receptor KIR3DL2 as a valuable diagnostic and prognostic marker for the detection of the tumoral T cell burden of Sézary syndrome patients. However, the function of this receptor on the malignant T lymphocyte population remained unexplored. The specific expression of KIR3DL2 by SS patients malignant cells prompted us to investigate its possible influence on mechanisms regulating the tumoral cells outgrowth and apoptosis process.To this aim, two axes were developed. The first axis aimed to highlight the function of KIR3DL2 on the malignant T lymphocyte population and to elucidate the intracellular signaling mechanisms initiated by engagement of the receptor with the monoclonal antibody AZ158. Our results show that KIR3DL2 can exert an inhibitory co-receptor function in malignant Sézary cells. Indeed, triggering of KIR3DL2 inhibits the CD3-mediated proliferation and cell death of the CD4+ KIR3DL2+ cells, this inhibition being correlated to a down-modulation of the TCR-mediated signals. Thus, KIR3DL2 does not behave as an independent signaling unit in Sézary cells, unlike NK cells.The second axis aimed to evaluate a new function of KIR3DL2 as CpG ODN receptor. We show for the first time a direct effect of CpG ODN on tumoral CD4+ T Sézary cells. Thus, we observed a caspase-dependent apoptotic effect of CpG ODN-C on Sézary cell lines and circulating malignant T cells. This process of cellular death is correlated to a dephosphorylation of the transcription factor STAT3, which is found constitutively phosphorylated and activated in Sézary cells.This study has provided new insights into the function and the intracellular signaling pathways initiated by KIR3DL2 in malignant Sézary T cells. Furthermore, this work opens new therapeutic perspectives based on the direct and specific targeting of tumor cells that could be associated to immune cell stimulation through the use of ODN CpG
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Goranova, Irena [Verfasser]. "Die segmentspezifische Lokalisation von Kir4.1, THIK-1 und THIK-2 Kaliumkanälen in der Säugerniere / Irena Goranova." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2017. http://d-nb.info/1126504718/34.
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Timmermann, Aline [Verfasser]. "Functional impact of Kir4.1 channels in hippocampal NG2 glia on neuronal plasticity and behavior / Aline Timmermann." Bonn : Universitäts- und Landesbibliothek Bonn, 2020. http://d-nb.info/1219140368/34.
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Tsiaze, Ernest Beaudelaire Verfasser], Marjan [Akademischer Betreuer] Rupnik, Tobias [Akademischer Betreuer] [Moser, and Blanche [Akademischer Betreuer] Schwappach. "Age-dependent changes in the exocytotic efficacy in Kir6.2 ablated mouse pancreatic beta cells / Ernest Beaudelaire Tsiaze. Gutachter: Tobias Moser ; Blanche Schwappach. Betreuer: Marjan Rupnik." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://d-nb.info/1049825829/34.
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Todorov, Zlatomir. "Computational study of the structure-function relationship of Kir3 channels and applications to the design of light-gated Kir3 channels." Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV030.
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Cette thèse de doctorat porte sur une famille de protéines transmembranaires exprimées sous forme de tétramères qui fonctionnent en tant que canaux ioniques sélectifs pour le potassium - le membre 3 des canaux potassiques à rectification entrante (Kir3). Quatre gènes codant pour les protéines Kir3 sont présents chez les mammifères dans la sous-famille, à savoir les membres Kir3.1-4. L'activité et l'expression tissulaire varient fortement en fonction des sous-unités constituant les canaux Kir3. Ces canaux sont activés par des interactions simultanées et directes avec des dimères bêta-gamma de la protéine G (Gβγ) libérés par les hétérotrimères de la protéine G de la classe G(i/o), des lipides PIP2 du feuillet interne de la membrane et des ions Na+ intracellulaires. Ces canaux permettent la genèse d’un courant potassique hyperpolarisant à des potentiels membranaires proches de l'EK. Ils jouent un rôle important dans la régulation de l'excitabilité cellulaire dans différents tissus. Des maladies ont été associées à une mauvaise régulation des canaux Kir3 de type sauvage ou à des mutations de leurs gènes.Un de mes objectifs a été de caractériser la dynamique conformationnelle sous-jacente à la fonction du canal Kir3.2 en utilisant la dynamique moléculaire. En partant de modèles obtenus par cristallographie aux rayons X, au lieu d’utiliser Gβγ pour ouvrir le canal dans nos simulations nous avons cherché à établir la stoichiométrie d’un ensemble de petites molécules connues pour l’activation de Kir3.2. Ainsi, dans une série de trajectoires où différentes combinaisons de partenaires étaient modélisées, le canal Kir3.2 a traversé différents états conformationnels et conducteurs – tout en reproduisant des caractéristiques décrites dans la littérature. Cette approche a amené à la découverte de nouveaux sites d'interaction lipide-protéine, potentiellement impliqués dans un mécanisme de rétroaction négative intrinsèque au canal Kir3.2. Cette prédiction est corroborée par des données de cristallographie aux rayons X publiées antérieurement et non exploitées dans ce contexte. L’analyse des trajectoires a également montré de nouvelles voies allostériques reliant les sites d’interaction du ligand aux portes du canal. De plus, sur la base de la dynamique du canal de type sauvage, nous avons proposé des mécanismes moléculaires pour plusieurs mutants pathologiques caractérisés expérimentalement.Parallèlement au projet théorique, j’ai travaillé à l’élaboration de canaux Kir3 sensibles à la lumière pour l’optogénétique. L’utilisation de modèles par homologie a guidé la sélection de résidus du canal qui ont été mutés en cystéine. La présence de cystéines réduites est nécessaire pour le marquage des protéines exprimées dans la membrane des ovocytes de Xenopus laevis avec les ligands photosensibles que nous avons utilisés. Parmi les vingt mutants cystéines criblées, l’un d’entre eux a montré un phénotype modéré de photo-activation. Ce phénotype a pu être considérablement amélioré par une mutation additionnelle, nous permettant d’obtenir le premier canal Kir3 constitutivement inactif et activable par la lumière.La procédure de simulation éprouvée dans ce travail est particulièrement adaptée à l’étude de divers canaux Kir3 d’intérêt. Une perspective pour nos modèles serait leur utilisation dans la conception de composés visant des conformations spécifiques du canal Kir3.2 en l’absence de données expérimentales sur les états d’ouverture et de fermeture complètes. Enfin, le canal Kir3.4 photosensible pourrait trouver des applications en biologie synthétique en tant que diode contrôlée par la lumière. En outre, ce mutant pourrait être inoculé in vivo – il devrait rester silencieux après marquage et fournir des informations sur le rôle physiologique de l'augmentation des courants médiés par canaux Kir3 lorsqu'il est activé par la lumière ultraviolette<br>This doctoral thesis focuses on a family of transmembrane proteins expressed as tetrameric assemblies which function as selective potassium ion channels – the potassium inward rectifier member 3 (Kir3). In the sub-family, four genes coding Kir3 proteins are present in mammals, namely, members Kir3.1 4. The activity and tissue-specific expression of Kir3 channels greatly vary with subunit composition. Kir3 channels are activated by direct simultaneous interactions with G-protein beta-gamma dimers (Gβγ) released from G-protein heterotrimers of the G(i/o) class, PIP2 lipids from the inner membrane leaflet and intracellular Na+ ions. These channels mediate hyperpolarizing potassium currents at membrane potentials close to EK, thus they are critical for development and health as they play an important role in decreasing cell excitability in different tissues. Disease conditions have been linked to misregulation of wild-type Kir3 channels or mutations of their genes.Starting from an X-ray crystallographic model, our goal was to obtain trajectories demonstrating simulated molecular dynamics of the wild-type Kir3.2 channel. We optimized a computational protocol using a set of brute-force simulations where different combinations of Kir3 activators were implemented. We observed the Kir3.2 channel transitioning between different conformational and conductive states – successfully reproducing dynamic features reported in the literature. We discovered novel functional lipid-protein interaction sites potentially involved in a negative-auto-feedback mechanism intrinsic to the Kir3.2 channel. Interestingly, this prediction is backed by previously published X ray crystallography data not exploited in this scope. Detailed analysis of the trajectories provided description of novel allosteric pathways linking the channel gates and the ligand interaction sites. In addition, based on the observed dynamics of the wild-type channel, we proposed molecular mechanisms for several experimentally-characterized pathologic mutants.In parallel to the computational project, we attempted to engineer light-gated Kir3 channels. This work was aided by homology models which guided the selection of positions satisfying an arbitrary set of criteria for cysteine mutagenesis. The presence of reduced cysteines is required for the labeling of the proteins expressed into the membrane of Xenopus laevis oocytes with the photoswitchable tethered ligands (PTL) we used. Among the twenty screened cysteine mutants, one construct showed a mild photo-blocking phenotype. Further engineering greatly improved this phenotype, yielding the first light-activated Kir3.4 homotetrameric channel.We believe that our simulation procedure is particularly suited for the investigation of various Kir3 assemblies of interest. An additional perspective for our models is to be used for the rational design of compounds targeting a specific conformation of Kir3.2 channel in the absence of experimentally obtained fully open/closed and transition-states conformations. Lastly, the developed photo-blocked Kir3.4 homotetramer could find applications in synthetic biology as a light-enabled diode. Alternatively, this mutant could be knocked-in in vivo – it should remain silent upon PTL-labeling in the resting-state, and provide information on the physiological role of the increase of Kir3 mediated currents when activated by ultra-violet light
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Niescierowicz, Katarzyna. "Développement de la technologie des récepteurs couplés à un canal ionique pour des études structure-fonction des récepteurs couplés aux protéines G et du canal Kir6.2." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-01067669.
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Les Récepteurs Couplés à un Canal Ionique (ICCRs) sont des canaux ioniques artificielscréés par fusion d'un Récepteur Couplé aux Protéines G (RCPG) au canal ionique Kir6.2. Dansce concept, le canal agit comme un rapporteur direct des changements conformationnels desRCPGs permettant de détecter par simple mesure de courant, la fixation d'agonistes etd'antagonistes proportionnellement à leur concentration.Le signal induit étant directement corrélé à l'activité du récepteur, indépendamment desvoies de signalisation des protéines G, nous avons exploité cet avantage pour étendre le champd'applications des ICCRs au cours de cette thèse. Nous avons développé quatre applications quisont: 1) la caractérisation fonctionnelle des RCPG optimisés pour la cristallisation par insertionde domaine du lysozyme du phage T4 dans la boucle ICL3; 2) la détection de la dépendance desRCPGs au cholestérol; 3) la détection de ligands dits "biaisés" pour faciliter leur criblage; et 4) lacartographie fonctionnelle des portes du canal Kir6.2 régulées par des protéines membranairesinteragissant par le domaine N-terminal.
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Ryan, Devon P. "Characterization of a novel inwardly rectifying potassium channel (Kir2.6) and its role in thyrotoxic hypokalemic periodic paralysis." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3398884.
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Fuchs, Lorenz. "Interaktion von Kir2-Kanälen mit 7-Helix-Rezeptoren." kostenfrei, 2009. http://nbn-resolving.de/urn/resolver.pl?urn=nbn:de:bvb:20-opus-39000.
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Rogalski, Sherri Lynn. "Modulation of Kir3 by lipids and tyrosine phosphorylation /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/6274.
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Bétourné, Alexandre. "Etude pharmacologique de la synapse Fibres Moussues / CA3 : rôles de la dynorphine, du zinc et des canaux KATP KIR6.2/SUR1 dans la mémoire contextuelle chez la souris." Toulouse 3, 2008. http://thesesups.ups-tlse.fr/419/.
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Cette thèse analyse l'implication de plusieurs éléments de la synapse Fibres Moussues (FM) / CA3-hippocampique dans la formation de la mémoire contextuelle chez la souris. La région CA3 de l'hippocampe est un véritable réseau auto-associatif qui participe à la création de souvenirs complexes, de type épisodique. Les FM, principales projections des cellules granulaires du gyrus denté sur les neurones du CA3, sont essentielles aux processus d'apprentissages et de mémorisation. Elles sont capables de libérer, en plus du glutamate (leur neurotransmetteur principal), de nombreux neuromodulateurs. 1- Les FM co-libèrent un peptide opioïde, la dynorphine, dont les récepteurs cibles, les récepteurs opioïdes Kappa (KOR), sont localisés sur les terminaisons présynaptiques des fibres. Par des injections pharmacologiques localisées dans la région CA3 d'agonistes et/ou d'antagonistes sélectifs, nous avons montré que l'activation pharmacologique des KOR immédiatement après un apprentissage dans le paradigme de peur conditionnée au contexte (CPC), supprimait la mémoire contextuelle chez la souris. Les KOR sont également une des nombreuses cibles modulées par le zinc vésiculaire, un neurotransmetteur atypique co-localisé avec le glutamate dans des vésicules spécifiques, et libéré par les FM. 2- Les travaux antérieurs de notre laboratoire ont montré que la chélation pharmacologique du zinc hippocampique perturbait l'acquisition et la consolidation de la mémoire contextuelle (Daumas et al. , 2004). Afin de répondre aux problèmes de spécificité posés par les chélateurs du zinc et de clarifier les rôles joués par cet ion, nous avons essayé d'invalider le gène codant le transporteur ZnT3, une protéine internalisant le zinc vésiculaire dans cette région. L'injection intra-tissulaire ou intracérébroventriculaire de siRNAs nus visant l'ARNm de ce transporteur n'a pas eu d'effets sur le comportement et les concentrations hippocampiques en zinc. Nous avons donc commencé à développer un protocole original d'administration des siRNAs: l'électropulsation in situ. 3- Finalement, nous avons étudié l'implication des canaux KATP Kir6. 2/SUR1 de la région CA3 dans la mémoire contextuelle. Ces canaux possèdent un site de liaison spécifique pour le zinc et ils participeraient au contrôle métabolique de la neurosécrétion des FM. .<br>This work analyses the putative involvement of several actors of the mossy fibers/CA3 synapse in the processing of contextual memory in mice. The CA3 of hippocampus forms a structured autoassociative network thought to process complex learning and episodic-like memories. Mossy fibers (MF), the main excitatory projections from dentate gyrus granule cells to the CA3, are essential for driving the storage of informations. MF terminals contain high amounts of vesicular zinc co-released with glutamate. Zn2+ has been shown to play the role of an atypical neurotransmitter in the hippocampus and might be required for the processing of memory at MF/CA3 synapses. 1- The hippocampus contains high amounts of Dynorphin, an opioid peptide co-released with glutamate from mossy fiber synapses. We performed transient pharmacological modulation of MF presynaptic Kappa opioid receptors (KOR) using bilateral microinjections in the CA3. Single injections of a selective agonist, antagonist or both, were performed immediately after conditioning C57BL/6J mice in a fear conditioning paradigm (FC). The agonist specifically decreased context-induced response suggesting that CA3-KOR are involved in the early consolidation of contextual memory processing. Importantly, among many other targets, KOR are sensitive to zinc modulation. 2- Previous works in our laboratory have shown the involvement of zinc released by MF in contextual memory (Daumas et al. , 2004). In order to improve our knowledge on the role played by MF-Zn2+ in memory, we injected specific naked siRNAs targeting the ZnT3 transporter, a protein internalizing zinc in MF synaptic vesicles. Repeated injections of different siRNAs either in the dentate gyrus or in the lateral ventricles were nearly without effects on mice behaviour in the FC and failed to alter hippocampal zinc levels. Meanwhile, we adapted the well known electropulsation technique in order to introduce nucleic acids in restricted brain areas of adult mice under brief anaesthesia. We will apply this technique for the delivery of anti-ZnT3 siRNAs in future experiments. 3- Finally, we evaluated the involvement of hippocampal ATP-sensitive potassium Kir6. 2/SUR1 channels (KATP) in learning and memory. .
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Hattori, Tetsuhisa. "A novel gain-of-function KCNJ2 mutation associated with short QT syndrome impairs inward rectification of Kir2.1 currents." Kyoto University, 2012. http://hdl.handle.net/2433/157461.
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Ng, Keat-Eng. "The distribution and function of the ATP-sensitive potassium channel subunit Kir6.1 in cardiac and skeletal muscle cell lines." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444888/.
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ATP-sensitive potassium channels (KAtp) are present in the plasma membrane of a number of tissues but are also present on endomembranes such as the endoplasmic reticulum (ER) and mitochondria. They are involved in a number of physiological and pathophysiological processes and form a link between cellular metabolism and membrane excitability. Ischaemic preconditioning describes the phenomenon in which a short period of ischaemia protects against a more prolonged one. The ability of potassium channel openers such as pinacidil and nicorandil can mimic this phenomenon, with inhibitors such as glibenclamide to abolish this response, led to the suggestion that the final effector in this process was the sarcolemmal Katp channel as it was able to shorten the cardiac action potential reducing the energy requirements of the cell. However, a number of pharmacological observations were not compatible with this hypothesis as diazoxide, which does not activate the sarcolemmal channel, was able to mimic preconditioning. The focus of research then turned to the potential involvement of a KAtp channel present in the mitochondrial inner membrane called the mitoKArp channel. The molecular identification of this channel would be important and there is controversial evidence to suggest that Kiro.l may be a major component of the mitoKATP channel. I examined the hypothesis that the localisation of Kir6.1 is functionally significant in cardiac and skeletal muscle because it generates important K+ flux in intracellular membranes such as the ER and perhaps mitochondria. Co-localisation studies showed that transfected Kiro.l was located in the ER with a small but significant proportion in mitochondria. However, Kir6.1 was ER retained and not trafficked to the plasma membrane when co-expressed with its regulatory subunit, the sulphonylurea receptor SUR1. Immunofluorescent staining also detected the presence of endogenous Kir6.1 in these cell lines using antibodies specific to Kir6.1. The distribution of Kir6.1 suggests that it may play a role in reactive oxygen species (ROS) production, calcium (Ca) handling in the ER and perhaps cellular respiration in mitochondria. ROS production is often associated with KAtp channel opening and protection against cell death at reperfusion. My results showed that diazoxide induced ROS production in C2C12, HepG2 and HEK293 cell lines with glibenclamide abolishing this effect. However, in the absence of Kir6.1, the same response was still observed. This suggests that Kir6.1 is not involved in the mechanism that is responsible for ROS production. The functional role of KAtp channels were also examined in mitochondria by measuring flavoprotein and NADH autofluorescence, an index of mitochondrial redox state and mitochondrial membrane potential (Au/m) in C2C12 cells and rat ventricular myocytes. In myocytes, flavoprotein oxidation increased when cells were treated with 3-nitroproprionic acid (3-NPA) and diazoxide. Glibenclamide did not reverse this effect. However, this phenomenon was absent in C2C12 cells. Given these observations, 3-NPA and diazoxide did not affect the Avj/m in C2C12 cells whereas glibenclamide caused mitochondrial depolarisation. The Aij/m could not be measured in myocytes. A large proportion of Kir6.1 resides in the ER and I examined whether Kir6.1 would alter ATP-induced Ca2+ transients. Upon ATP stimulation, C2C12 cells released Ca2+ from internal stores via the P2Y purinergic signalling pathway. The use of dominant negatives (DN) for Kir6.1 showed that ATP-induced Ca2+ transients were affected by the absence of Kir6.1. However, on closer inspection it was revealed that the presence of eGFP to identify transfected cells seriously perturbed the Fura-2 signal. In conclusion, the KAtp sensitive channel subunit Kir6.1 is predominantly distributed in the ER with a small but significant proportion in mitochondria. I also report that pharmacological compounds such as diazoxide and glibenclamide are not always truly 'selective' for the activation and inhibition of KAtp channels. I have not identified a specific role for Kir6.1 but my data suggests that Kir6.1 is not part of the mitoKATp channel and Kir6.1 is not involved in ROS production and mitochondrial function but it may still have a role in Ca2+ handling.
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Ortonne, Nicolas. "Caractérisation phénotyque et fonctionnelle de l'expression de CD158k/KIR3DL2 à la surface des cellules néoplasiques du syndrome de Sézary." Paris 7, 2008. http://www.theses.fr/2008PA077056.
Full textAbstract:
Le syndrome de Sézary (SS) est un lymphome T cutané qui se caractérise par la prolifération dans le sang et la peau de lymphocytes T néoplasiques CD4+, appelés cellules de Sézary (CS). Nous avons montré que les CS expriment le récepteur p140/CD158k/KIR3DL2, de la famille des KIR (killer immunoglobulin-like receptor). L'expression membranaire de ce récepteur permet de caractériser les clones lymphocytaires T néoplasiques présents dans le sang des malades, identifiés par la technique de l'immunoscope, et de les distinguer de clones réactionnels. Dans la peau, l'expression transcriptionnelle de KIR3DL2 analysée par RT-PCR quantitative permet de faire le diagnostic différentiel avec les dermatoses inflammatoires érythrodermiques, et de caractériser la présence de cellules néoplasiques résiduelles dans la peau au stade débutant ou lors de rémissions partielles sous traitement. Si KIR3DL2 est le seul récepteur KIR exprimé de façon récurrente, d'autres KIR peuvent être exprimés par les CS. Nous avons montré dans la lignée de CS P1 que ces récepteurs sont fonctionnels, et que l'engagement de récepteurs activateurs (KIR2DS2 et 2DS3) favorise la prolifération cellulaire associée à la stimulation du CD3, en activant la voie de JNK, via une molécule adaptatrice encore non identifiée, alors que le récepteur inhibiteur KIR3DL2 l'inhibe. En l'absence d'anomalies génomique récurrente portant sur le locus des KIR, en position 19q13. 4, les mécanismes moléculaires conduisant à l'expression de récepteurs KIR par les CS restent inconnus. De même, le rôle de ces récepteurs, notamment de KIR3DL2, en terme de résistance à l'apoptose des cellules néoplasiques, reste à évaluer<br>Sezary syndrome (SS) is a cutaneous T-cell lymphoma characterized by the proliferation in the skin and blood of neoplastic T CD4+ lymphocytes, usually named Sezary cells (SC). We have shown that SC express the p140/CD158k/KIR3DL2 receptor of the KIR (killer immunoglobulin-like receptor) family. The cell surface expression of KIR3DL2 allows to characterize the malignant T-cell clones in the blood of patients, previously identified by the analysis of TCR Vbeta transcripts expression (immunoscope technique), and to differentiate them from reactive clones. In the skin, analysis of KIR3DL2 transcripts expression with quantitative RT-PCR allows to distinguish between SS and benign erythrodermic inflammatory dermatoses, and to identify the presence of neoplastic cells at early phases of the disease or at the time of partial remission, in the absence of circulating SC. Although KIR3DL2 appears to be the sole KIR receptor to be recurrently expressed by SC, other KIR can be present at their surface. We have shown in the SS cell line P1 that these receptors are functional, and that engagement of activatory forms (KIR2DS2 et 2DS3) enhances cell proliferation triggered by CD3 stimulation, by the recruitment of the JNK pathway through a yet unknown adaptatory molecule, whereas KIR3DL2 acts as an inhibitory receptor. In the absence of known genomic abnormality of the 19q13. 4 KIR locus, the molecular mechanisms underlying the expression of KIR by SC remains to be clarified. Further, the role of KIR, especially KIR3DL2, in the résistance of CS to apoptosis needs to be investigated
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Pini, Jonathan. "Modélisation du syndrome d'Andersen dans les cellules souches pluripotentes induites : implication du canal potassique Kir2.1 dans la morphogenèse osseuse." Electronic Thesis or Diss., Nice, 2016. http://theses.unice.fr/2016NICE4042.
Full textAbstract:
Le syndrome d’Andersen est une maladie rare et associée à la perte de fonction du canal potassique Kir2.1. Afin d’étudier sa physiopathologie, nous avons généré et caractérisé des cellules souches pluripotentes induites (iPS) contrôle et Andersen. Nous avons ensuite différencié ces cellules iPS en cellules souches mésenchymateuse (MSC). Les cellules MSC de patients présentent une capacité de différenciation en ostéoblastes et en chondrocytes diminuée par rapport aux cellules contrôle. En effet, la production de matrice extracellulaire et l'expression des master gènes des différenciations osseuses et cartilagineuses, est réduite chez les patients. Ces travaux de thèse montrent que le canal Kir2.1 est essentiel au développement osseux. Les défauts de différentiation observés pourraient expliquer les dysmorphies associées avec le syndrome d’Andersen<br>Andersen's syndrome is a rare disorder associated with a Kir2.1 potassium channel loss of fuction. To study the pathophysiology, we have generated and characterized induced Pluripotent Stem cells (iPS) from control and patient cells. We have then differentiated those iPS cells into mesenchymal stem cells (MSC). Patient's MSc have a lower osteoblastic and chondrogenic differnciation ability compared to control cells. Indeed, extracellular matrix production and master gene expression of osteoblastic and chondrogenic differenciation are reduced in patient’s cells. Alltogether, these results shown that Kir2.1 channel is required for bone developement. The differenciation defects saw in patient cells could explain the Andersen's syndrome associated dysmorphies
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Bignon, Yohan. "Physiologie et physiopathologie des transports transépithéliaux du tubule proximal : mise en évidence du rôle de la sous-unité Kir4.2 et analyse d'un mutant de ClC-5 impliqué dans la maladie de Dent." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066244/document.
Full textAbstract:
Le tubule proximal participe à la diurèse en modifiant la composition de l'ultrafiltrat glomérulaire. Grâce à de nombreux transports transépithéliaux, il le glucose, les acides aminés et les protéines de bas poids moléculaires, ainsi que 80 % des ions HPO42- ou HCO3-, 60 % des ions Na+, Cl-, K+, Ca2+, 75 % de l’eau et 30 % des ions Mg2+ ultrafiltrés.Durant ma thèse, j'ai étudié les rôles physiologiques et physiopathologiques de deux protéines de transport exprimées dans le tubule proximal.Dans le cadre de ma première étude, j'ai évalué in vivo la fonction rénale de souris n'exprimant pas une protéine appelée Kir4.2, dont le rôle est inconnu. Nos résultats montrent que Kir4.2, associée à Kir5.1, forme un canal potassique basolatéral Kir4.2/Kir5.1 dans le tubule proximal. L'absence de Kir4.2 provoque chez la souris une acidose tubulaire proximale isolée, consécutive à une ammoniogénèse altérée. De fait, la perte de fonctionnalité de Kir4.2 pourrait être à l'origine d'acidoses tubulaires proximales isolées familiales idiopathiques.Dans le cadre de ma seconde étude, j'ai analysé in vitro la fonctionnalité d'un mutant pathogène de l'échangeur 2Cl-/H+ ClC-5 impliqué dans la maladie de Dent. Cette maladie, caractérisée par une protéinurie de bas poids moléculaire associées à divers troubles du tubule proximal, serait liée à un défaut d'acidification des endosomes précoces par ClC-5. Toutefois, le mutant de ClC-5 que nous avons étudié, converti en canal chlorure, acidifie autant les endosomes précoces que le ClC-5 sauvage. Surprenants, ces résultats suggèrent que la maladie de Dent puisse être causée par un défaut d'accumulation d'ions chlorure dans l'endosome<br>The proximal tubule is involved in diuresis by modifying the content of the glomerular ultrafiltrate. Using a variety of transepithelial transports systems, it reabsorbs all ultrafiltrated glucose, amino-acids and low molecular weight proteins, as well as 80% of HPO42- and HCO3- ions, about 60% of Na+, Cl-, K+, and Ca2+ ions, 75% of water and 30% of Mg2+.During this thesis, I determined the physiological and physiopathological roles of two transport proteins present in proximal tubule. Firstly, I evaluated the renal function of mice invalidated for the Kir4.2 protein, whose role was undetermined. Our results show that Kir4.2, in association with Kir5.1, form a Kir4.2/Kir5.1 potassium channel at the basolateral membrane of proximal tubular cells. Furthermore, Kir4.2-null mice exhibit a reduced ammoniagenesis leading to an isolated proximal renal tubular acidosis. This study provides the gene encoding Kir4.2 as a candidate gene for the yet unexplained autosomal dominant isolated proximal renal tubular acidosis.Secondly, I evaluated in vitro the functional consequences of a pathogenic mutation of the 2Cl-/H+ exchanger ClC-5, involved in Dent’s disease. This disease, characterized by a low-molecular-weigth-proteinuria in the context of a general proximal tubule dysfunction, is currently thought to be due to an acidification defect of early endosomes linked to a loss of function of ClC-5. Surprisingly, our results show that ClC-5, converted into a chloride channel by this mutation, indeed acidifies the early endosomes as well as the ClC-5 wild-type. Thus, Dent’s disease may originate from a defect in the accumulation of chloride ions into the early endosomes
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Banas, Krystyna Anna. "K(ATP) channel Kir62 subunit distribution differs between muscles and between fiber types in skeletal muscle." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28483.
Full textAbstract:
The activity of the ATP-sensitive potassium (KATP) channel, whose sensitivity to ATP and ability to permit K+ flux together allow the channel to couple the metabolic state of a cell to its membrane excitability, is important in several tissues for the maintenance of glucose homeostasis and cytoprotection. In skeletal muscle specifically, the channel has been shown to be involved in cell volume regulation, modulation of glucose uptake, and the prevention of fiber damage and contractile dysfunction during fatigue. The extent of the cytoprotective capabilities of the KATP channel vary tremendously between muscles with different muscle fiber type composition. Semi-quantitative measurement of Kir6.2 subunit content showed that variances exist in KATP channel content between fiber types and between different muscles. These differences may be related to the extent of importance of the channel's function in a specific muscle. The highest Kir6.2 content was observed in the most glycolytic fiber types and in the more glycolytic muscles studied, and it is these muscles and fiber types which appear to be most dependent on functional KATP channels for their cytoprotective role.
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Tham, Daniel Kai Long. "ECM-receptor interactions regulate the distribution of Kir4.1 and AQP4 channels in renal tubules and at the blood-brain barrier." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44213.
Full textAbstract:
Osmostasis relies on the efficient and selective transport of molecules across barriers, often in a directional manner and this, in turn, is dependent on the asymmetric arrangement of channels within the cells comprising the organs that regulate these processes. The primary aim of this thesis is the identification of the extracellular matrix-receptor interactions important in the generation of polarized channel distribution in two such organs, the kidney and the blood-brain barrier. In the first study, we investigated the involvement of fibronectin and laminin in the basolateral localization of Kir4.1, an inwardly-rectifying potassium channel, and aquaporin-4 (AQP4), a water-permeable channel, in polarized Madin-Darby canine kidney (MDCK) cells. We determined using a variety of approaches that laminin-1 and fibronectin, when present in the culture substrate, significantly stablize both channel types at the basolateral surfaces of these cells and accordingly induce their increased expression within this membrane domain without requiring a concomitant upregulation of de novo channel synthesis. We also show that the coexpressed laminin receptor dystroglycan (DG) is important for cell surface expression of Kir4.1 but not AQP4, and demonstrate via the use of disintegrin peptides and function-blocking antibodies that their cell-surface expression and stability is also partly reliant on integrin receptors, with αvβ3 being particularly important in the case of the latter. In the second study, we examined the possibility that laminin-dystroglycan binding is involved in the regulation of AQP4 turnover in astrocytes. We first determined that laminin, when applied to primary astrocytes in culture, causes AQP4 amounts at the plasma membrane to increase in a DG-dependent manner while depleting the channel from intracellular sites, brought about by the suppression of channel endocytosis. We then showed that DG binds to inactive dynamin, and that the latter, when freed from this inhibitory influence, functions in cooperation with clathrin to mediate the rapid internalization of AQP4. Finally, we demonstrated that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4 only. Our findings therefore reveal that, through their roles in establishing the microscopic architecture of these systems, the extracellular matrix and cell-surface receptors are critical determinants in osmoregulation