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Structural determinants of regulation of surface delivery of NMDA receptors in mammalian cells
Danačíková, Šárka ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...
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Characterization of pore opening relevant residues in TM3 and TM4 domains of Orai1
ANDOVA, Ana-Marija
Calcium (Ca2+) ions play a crucial role in almost every aspect of cellular life. The most prominent calcium entry pathway into the cell is the calcium release-activated calcium (CRAC) channel, composed of the Orai1 protein, and the stromal interaction molecule STIM1. The channel is activated through conformational changes upon STIM1 coupling to the C-terminus of Orai1 protein following store depletion, which in turn allows Ca2+ influx into the cell. The abnormal function of the CRAC channel caused by mutations gives rise to distinct pathologies. Since it has not yet been elucidated how the signal propagation moves to the pore upon coupling, this thesis dives into its investigation by focusing on characterizing the TM3 and TM4 domains and their importance in leading to an open permissive conformation of the channel. The pivotal foundation for the creation of novel strategies in the modulation of the Orai1 function lies with the understanding of the dynamics of the Orai1 pore opening.
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Structural determinants of regulation of surface delivery of NMDA receptors in mammalian cells
Danačíková, Šárka ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...
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Impact of protein transmembrane domains on membrane organisation
Šljivnjak, Erna ; Cebecauer, Marek (advisor) ; Novotný, Marian (referee)
Plasma membrane is not a static, but rather a dynamic structure that constantly changes its form and local properties. Interactions between building blocks of plasma membrane and external factors are responsible for those changes. In this thesis, I summarize the literature which describes interactions between transmembrane proteins and lipid membranes as well as its consequences. I discuss membrane thickness, respectively thinning, protein sorting and clustering shown to be dependent on the properties of transmembrane domains. Furthermore, the role of proteins in various model of the plasma membrane organization are indicated. Finally, I report on currently discovered impact of the surface roughness of TMDs on local mobility and organization of lipids. All these data indicate importance of detailed understanding of TMDs, their properties and relation to surrounding lipid membranes. Key words: membrane, protein, transmembrane domain, hydrophobic mismatch, protein sorting, models of the plasma membrane
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