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Functional and pharmacological properties of GluN1/GluN2 and GluN1/GluN3 subtypes of NMDA receptors
Kolcheva, Marharyta ; Horák, Martin (advisor) ; Bohačiaková, Dáša (referee) ; Balaštík, Martin (referee)
(EN) N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors and they play a critical role in excitatory synaptic transmission in the mammalian central nervous system (CNS). Hyperactivity or hypoactivity of NMDARs can lead to a wide spectrum of pathological conditions and psychiatric disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, schizophrenia. NMDARs form a heterotetrameric complex made up of GluN1, GluN2(A-D) and/or GluN3(A, B) subunits. Different subtypes of NMDARs could have various effects on disease pathogenesis and therefore it is crucial to investigate the specific role of each subunit in the regulation of normal NMDAR functioning. The regulation of NMDARs occurs at different levels, from early processing, including synthesis, assembly, quality control in the endoplasmic reticulum (ER), trafficking to the cell surface, to internalization, recycling, and degradation. In this dissertation, we mainly focused on determining the roles of extracellular and transmembrane regions of different subtypes of NMDARs in the regulation of their function. In particular, using electrophysiology and microscopy methods on HEK293 cells and cultured hippocampal neurons, we investigated: (i) the impact of N-glycosylation and different lectins on...
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Identification of new neuroactive steroids that are able to interact with allosteric binding sites on purinergic P2X receptors
Sivčev, Sonja ; Zemková, Hana (advisor) ; Vyklický, Vojtěch (referee) ; Stojilkovic, Stanko S. (referee)
(EN) Purinergic P2X receptors are ATP-gated cation channels with multiple physiological roles and are emerging as important therapeutic targets in a range of diseases. P2X subunit consists of two transmembrane helices (TM1 and TM2), an extracellular ATP-binding domain, and intracellular N- and C- termini. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. P2X are ubiquitously expressed. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain. The activity of P2X depends not only on the presence of ATP but also on allosteric modulators that may inhibit or potentiate the activity of these channels. Our aim was to identify new molecules that could interact with allosteric binding sites on P2X receptors, design and synthesize new analogues of neurosteroids, and define crucial receptor domains and amino acids important for neurosteroid binding. By using a patch-clamp electrophysiology technique we recorded ATP-induced currents in HEK293T cells transfected with rat P2X2, P2X4, and P2X7, as well as in the rat anterior pituitary cells and hypothalamic neurons endogenously expressing these receptors. We found that 17β-ester derivatives of testosterone, namely testosterone butyrate and...
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Contribution of ten ectodomain cysteine residues to function of ATP-gated P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Teisinger, Jan (referee)
Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...
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Transport studies in vitro on 2D and 3D cellular level
Urbanová, Johana ; Ramos Mandíková, Jana (advisor) ; Smutná, Lucie (referee)
Farmaceutická fakulta v Hradci Králové Studentka: Johana Urbanová Školitelka: PharmDr. Jana Ramos Mandíková, Ph.D. Název diplomové práce: Transportní studie na 2D a 3D buněčné úrovni. Trojrozměrné (3D) buněčné modely lépe napodobují buněčné podmínky in vivo než tradičně používané dvourozměrné (2D) buněčné modely. Transportéry pro organické anionty (OATs) a kationty (OCTs) hrají důležitou úlohu v ledvinné eliminaci léčiv a mají vliv na jejich farmakokinetické vlastnosti. Cílem práce bylo vytvořit 2D a 3D buněčný model z embryonálních ledvinných buněk HEK293, oba modely přechodně transfekovat transportérem hOAT1, transfekci optimalizovat a provést transportní inhibiční studie s vybranými NSAIDs (diklofenakem, ibuprofenem, indometacinem a naproxenem). 2D buněčný model jsme vytvořili ve 24jamkové destičce ve formě monovrstvy, jako 3D buněčný model jsme používali sféroidy, které jsme zhotovili na Petriho misce metodou visící kapky. Při transportních studiích jsme buňky inkubovali s radioaktivním substrátem OATs p-aminohippurovou kyselinou ([3 H]PAH) s nebo bez příslušného inhibitoru. Inhibiční vliv NSAIDs jsme porovnávali pomocí parametru IC50. 2D i 3D model z buněk HEK293 se nám povedlo vytvořit, ale následná transfekce byla úspěšná pouze v monovrstvě buněk. Funkčnost 3D modelu se pomocí transportní studie...
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína ; Horák, Martin (advisor) ; Novotný, Jiří (referee) ; Valeš, Karel (referee)
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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Transport studies in vitro on 2D and 3D cellular level
Urbanová, Johana ; Ramos Mandíková, Jana (advisor) ; Smutná, Lucie (referee)
Farmaceutická fakulta v Hradci Králové Studentka: Johana Urbanová Školitelka: PharmDr. Jana Ramos Mandíková, Ph.D. Název diplomové práce: Transportní studie na 2D a 3D buněčné úrovni. Trojrozměrné (3D) buněčné modely lépe napodobují buněčné podmínky in vivo než tradičně používané dvourozměrné (2D) buněčné modely. Transportéry pro organické anionty (OATs) a kationty (OCTs) hrají důležitou úlohu v ledvinné eliminaci léčiv a mají vliv na jejich farmakokinetické vlastnosti. Cílem práce bylo vytvořit 2D a 3D buněčný model z embryonálních ledvinných buněk HEK293, oba modely přechodně transfekovat transportérem hOAT1, transfekci optimalizovat a provést transportní inhibiční studie s vybranými NSAIDs (diklofenakem, ibuprofenem, indometacinem a naproxenem). 2D buněčný model jsme vytvořili ve 24jamkové destičce ve formě monovrstvy, jako 3D buněčný model jsme používali sféroidy, které jsme zhotovili na Petriho misce metodou visící kapky. Při transportních studiích jsme buňky inkubovali s radioaktivním substrátem OATs p-aminohippurovou kyselinou ([3 H]PAH) s nebo bez příslušného inhibitoru. Inhibiční vliv NSAIDs jsme porovnávali pomocí parametru IC50. 2D i 3D model z buněk HEK293 se nám povedlo vytvořit, ale následná transfekce byla úspěšná pouze v monovrstvě buněk. Funkčnost 3D modelu se pomocí transportní studie...
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína ; Horák, Martin (advisor) ; Novotný, Jiří (referee) ; Valeš, Karel (referee)
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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