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On the role of the first transmembrane domain in desensitization kinetics of the P2X4 receptor.
Kalasová, Ilona ; Zemková, Hana (advisor) ; Krůšek, Jan (referee)
Extracellular adenosin-5'-triphosphate (ATP) is an important signalling molecule. Cells of eukaryotic tissues release ATP and express responding purinergic receptors. Ionotropic P2X receptors are trimeric ion channels permeable for K+, Na+ and Ca2+ ions. Each subunit consists of two transmembrane domains (TM1 and TM2), an extracellular loop and intracellular N- and C- termini. The transmembrane region is formed by six helical domains. According to the known crystal structure of zfP2X4 receptor, TM1 helixes are oriented peripherally and stabilize TM2 helixes which form the ion gate. However, eletrophysiological studies revealed that TM1 might also participate in channel gating and forming of the ion pore in the open state. The aim of this work was to investigate the role of TM1 in the process of desensitization of rat P2X4 receptor using cystein-scanning mutagenesis. Mutation of two residues (in Asn32 and Tyr42) prolonged desensitization of P2X4 receptor. Moreover, experiments with a partial agonist α,β-methylenadenosin-5'-triphosphate (αβ-meATP) proved that conformation change of TM domains in the process of desensitization is independent on conformation change caused by an agonist binding. Conserved residue Tyr42 is located in the proximity of TM2 of neighbouring subunit. It probably interacts with Met336...
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Functional role of purinergic P2X receptors in the supraoptic nuclei of the rat and structure-function relations of recombinant P2X receptors.
Vávra, Vojtěch ; Zemková, Hana (advisor) ; Novák, František (referee) ; Vyklický, Ladislav (referee)
Purinergic P2X receptors are non-selective cationic channels gated by extracellular ATP. Up to now, seven mammalian subunits, termed P2X1-X7, have been cloned and characterized. These receptors comprise a new membrane channel family with distinct structural and functional features. P2X receptors take part in a signalling network called "purinergic signalling" which is widely exploited in both somatic and neuronal tissues. In the central nervous system, they are highly expressed in the hypothalamus and hypophysis, where they participate in the regulation of homeostatic and reproductional functions. The main focus of my Thesis is on the expression and functional role of P2X receptors in supraoptic nuclei of the rat hypothalamus. These nuclei contain two populations of magnocellular neurons which produce either oxytocin or arginine-vasopressin. Delivery of the hormones into the systemic blood relies on the electrical activity of supraoptic neurons, which is in turn governed by the incomming synaptic inputs. It has been recently shown, that the process of hormone release from supraoptic neurons is regulated by extracellular ATP. However, purinergic signals that regulate hormone secretion are not well understood. The aim of my study was to identify subtypes of P2X receptors expressed in the supraoptic...
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Astrocyte volume changes during brain ischemic injury
Mikešová, Michaela ; Zemková, Hana (referee) ; Anděrová, Miroslava (advisor)
Brain ischemic injury is a complex of pathophysiological events following transient or permanent reduction of brain blood flow. It results in a disruption of neuronal and astrocytic physiological functions, long-term reduction of brain blood flow leads to the cell death. Number of recent studies is focused on astrocytes, which might play key roles in surviving cells, including neurons, during ischemic injury. Astrocytes provide many important functions, such as maintenance of ionic homeostasis, prevention of excitotoxicity, scavenging free radicals and others and thus astrocytes may dramatically swell during ischemic conditions and contribute notably to the development of cytotoxic edema. This thesis summarizes mechanisms possibly contributing to the astrocytic swelling during brain ischemic injury as well as methods used for studying astrocyte volume changes and their quantification. Since the brain edema dramatically complicates both course and treatment of ischemic injury, knowledge of mechanisms leading to astrocytic swelling and their volume regulation during ischemia/reperfusion might be used for developing new therapeutic approaches for the treatment of cerebral ischemia, mainly for reduction of negative impact of edema.
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Contribution of ten ectodomain cysteine residues to function of ATP-gated P2X4 receptor
Tvrdoňová, Vendula ; Teisinger, Jan (referee) ; Zemková, Hana (advisor)
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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The role of transmembrane domains in the structure and function of P2X receptors
Jindřichová, Marie ; Zemková, Hana (advisor) ; Langmeier, Miloš (referee) ; Martásek, Pavel (referee)
Purinergic P2X receptors represent a novel structural type of ligand-gated ion channels activated by extracellular ATP. So far, seven P2X receptor subunits have been found in excitable as well as non-excitable tissues. In the past ten years, the number of studies on P2X receptors has dramatically increased as investigators have begun to determine the physiological roles played by extracellular ATP and specific P2X receptor subtypes. It is already known that purinergic signaling is a key mechanism in pain sensation, brain injury, and immune processes. Little is known about their structure, mechanism of channel opening, localization and termination of ATP action by ectonucleotidases. Detailed knowledge about these events and the structure of purinergic receptor proteins evoke hope that new drugs will be developed that could prevent chronic pain and would be effective in protection against many diseases. The aim of this work is to summarize recent investigations and describe our contribution to elucidating the structure of P2X receptors. We examined the structure of transmembrane domains of the P2X4 receptor subtype, the main purinergic receptor-channel in the central nervous system, the mechanism of channel opening and closing and its sensitivity to agonists and allosteric modulator ivermectin. To...
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Specifics of Entrepreneurial Environment, Business and Cultural Customs of the region Rhone-Alpes
Zemková, Hana ; Gullová, Soňa (advisor) ; Štemberk, Jan (referee)
The aim of my bachelor thesis is to analyse the entrepreneurial environment and outline specifics of business negotiation and cultural customs of the French region Rhône-Alpes and also to verify the theory that the region is suitable for entrance of Czech entrepreneurs. The paper could serve as a comprehensive guide for potencial investors, entrepreneurs and business representatives. The body of the thesis is divided into seven chapters. The introductory chapter provides basic characteristics of the region from the administrative, geographic, demographic and historic point of view. The second chapter analyses economical environment, industry, agriculture, infrastructure, tourism and foreign trade of the region. The content of the third chapter is evalution of technological environment that plays a very important role in the region. The fourth chapter is focused on business customs, etiquette and cultural specifics, whose knowledge is essential for successful business cooperation. The fifth chapter evaluates attractiveness of the entrepreneurial environment and names its strengths and weaknesses. The sixth chapter is dedicated to cooperation between the Czech Republic and the region. The last chapter contains questionnaire and results of questionnaire inquiry. The paper is based on Czech and foreign books and internet sources, statistics made by international organizations and is amended by information provided by Mr. Martin Dytrych from the French-Czech Business Chamber and personal experience of entrepreneurs.
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