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Characterization of native and heterologously expressed membrane transporters in yeast using fluorescent probes
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Cebecauer, Marek (referee) ; Krůšek, Jan (referee)
Yeast plasma membrane transporters play crucial roles in many cellular processes, including detoxification and build-up and maintenance of the plasma membrane potential (ΔΨ). The former development of the diS-C3(3) fluorescence assay by the Biophysics Group of the Institute of Physics, Charles University, enabled us to conveniently study both, including their changes, using a simple fluorescent probe diS-C3(3). Many studies carried out on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using the diS- C3(3) assay for multidrug-resistance pump inhibitors in a set of isogenic yeast pdr5 and snq2 deletion mutants we found that n-alcohols (from ethanol to hexanol) exhibit an inhibitory effect on both pumps, increasing with the length of the alcohol carbon chain. The inhibition is not connected with loss of plasma membrane structural or functional integrity and is fully reversible. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins. Tok1p is a highly specific...
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Study of membrane transport processes in yeast using potentiometric fluorescent porbe diS-C3(3)
Bartl, Tomáš ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
1 Title: Study of membrane transport processes in yeast using potentiometric fluorescent probe diS-C3(3) Author: Tomáš Bartl Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Dana Gášková, CSc., Institute of Physics of Charles University Abstract: Yeast membranes contain a number of transporters. Some are responsible for flow of nutrients to the inside of the cell, others for disposing of waste and foreign substances and some for transport of small ions or protons across the membrane. The focus of this work is on the activity of specific transport membrane proteins, so-called MDR pumps, which are responsible for transport of foreign substances or drugs, out of the cell. Using the series of mutant strains of the yeast Saccharomyces cerevisiae (AD1-3, AD1-8 and AD12) differentiated in the presence of specific MDR pumps in their membrane, an influence of various chemical substances on the intracellular concentration of the potentiometric fluorescent probe diS-C3(3), which is actively being transported out of the cell by some of the MDR pumps, was observed. By the examination of the effect of 2-deoxyglucose we proved the active contribution of not only the main MDR pump, Pdr5p, but also of some other pumps, in lowering the intracellular probe concentration. It was observed that...
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The role of spinal TRPV1 receptors in nociceptive signalling and the modulatory effect of chemokine CCL2 and µ-opioid receptor agonists
Šulcová, Dominika ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
The first nociceptive synapse in the spinal cord dorsal horn represents an important site, where nociceptive synaptic transmission can be modulated under pathological conditions. One of the modulatory mechanism involves activation of the transient receptor potential vanilloid 1 (TRPV1) that is expressed on central terminals of primary nociceptive neurons, where it regulates release of neurotransmitters and neuromodulators. Previous studies suggested that changes in TRPV1 activity may be related to effects of chemokine CCL2 (C-C motif ligand 2) and may be also involved in synaptic transmission modulation after µ-opioid receptors (MOP-R) activation. Because CCL2 receptors CCR2 often co-localize with TRPV1 and MOP-R, the goal of this work was to studypossible interactions of these receptors on the pre-synaptic endings of primaryafferents in the spinal cord dorsal horn and their role in nociceptive signalling under pathological conditions. The presented thesis focused on the effect of CCL2 during peripheral neuropathy and its interference with µ-opioid receptor activation. To studysynaptic transmission at the spinal cord level, patch-clamp recordings of excitatory post-synaptic currents (EPSC) in superficial spinal cord dorsal horn neurons in acute lumbar spinal cord slices from rats was used....
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The interaction of kainate subtypes of glutamate receptors with steroid compounds.
Fraňková, Denisa ; Krůšek, Jan (advisor) ; Adámek, Pavel (referee)
Kainate receptors belong to the family of glutamate receptors, which include NMDA, AMPA and δ receptors. Glutamate receptors are widely found in the brain and therefore they are very dynamically investigated, especially from view of pharmacology, because there is great potential for finding new and more specific modulators which could be used in the treatment of neurodegenerative diseases. The aim of this work was to extend the knowledge about the influence of neurosteroids on homomeric kainate receptors (GluK1, GluK2, GluK3) in which is the study of modulation by neurosteroids still at the beginning. We have investigated interactions of homomeric kainate receptors with selected neurosteroids (pregnenolone sulfate, pregnanolone sulfate, dehydroepiandrosterone, dehydroepiandrosterone sulfate) by using patch clamp method in the configuration of whole-cell recording and also by using microfluorometry. We have found out that the biggest modulating effect on homomeric kainate receptors is caused by pregnenolone sulfate, which inhibits glutamate responses of these receptors. Keywords kainate receptor, glutamate, neurosteroids, steroids, patch-clamp technique
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Role of the segment 400-500 in biological activity of Bordetella pertussis adenylate cyclase toxin
Suková, Anna ; Mašín, Jiří (advisor) ; Krůšek, Jan (referee)
The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in virulence of the whooping cough agent Bordetella pertussis. It translocates an AC enzyme into cytosol of CD11b+ phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. In parallel, CyaA permeabilizes cellular membrane by forming cation-selective pores. The goal of my diploma thesis was an analysis of the mechanism of interaction of the segment linking the invasive adenylate cyclase domain and the RTX hemolysin moiety of CyaA with target membrane. Our data show that the segment linking the AC to the hydrophobic domain of CyaA is directly involved in the interaction of the toxin with the membrane and controls the formation of small cationt-selective pores. Our results generate new knowledge that will be of relevance to the entire field of toxin biology and will enable the design of improved CyaA- based vaccines. Keywords: Bordetella pertussis, adenylate cyclase toxin, membrane translocation, pore- forming activity, black lipid bilayers, liposomes
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Identification of the binding sites on transient receptor potential cation channel TRPC6 for Calmodulin and S100A1
Bílý, Jan ; Teisinger, Jan (advisor) ; Krůšek, Jan (referee) ; Pavlíček, Jiří (referee)
Identification of the binding sites on transient receptor potential cation channel TRPC6 for Calmodulin and S100A1 The TRP (transient receptor potential) group of ion channels represents a large subset of membrane receptors. A part of this supergroup are canonical TRPC channels with a sequence homology analogical to TRP receptor first discovered at fruit fly (Drosophila melanogaster). These membrane channels are involved in a variety of physiological functions in different cell types and tissues. TRPC6 is a non-selective cation channel that modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. TRPC6 channel contains binding domain CIBR (Calmodulin inositol binding region), which is also able to adapt to calcium binding protein S100A1. Characterisation of the integrative binding site for calmodulin (CaM) and S100A1 at the C-tail of TRPC6 is presented in this work. Using site-directed mutagenesis, soluble protein fragments TRPC6 CT (801-787) were prepared with intentional changes in amino acid sequence. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by measurement of fluorescence anisotropy influence and their participation in the calcium-dependent binding of CaM and/or S100A1 to...
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Localization and characterization of binding sites for Ca2+ binding proteins and phosphatidylinositol phosphates on intracellular termini of TRP channels
Boušová, Kristýna ; Teisinger, Jan (advisor) ; Žáčková, Markéta (referee) ; Krůšek, Jan (referee)
This dissertation concerns with characterization of binding sites for calcium binding protein S100A1 and phosphatidylinositol phosphates on intracellular regions of transient receptor potential channels (TRPs), particular from canonical (TRPC), vaniloid (TRPV) and melastatin (TRPM) families. TRPs represent superfamily of important mediators that play critical roles in sensory physiology: contributions to taste, olfaction, vision, hearing, touch and thermo- and osmo- sensation. They serve as non-selective and nociceptive membrane receptors responsible for the modulation of driving force for cations entry into the cell. TRPs are composed from six transmembrane domains and N- and C- termini intracellular regions. Overall four monomer units form a characteristic assembly of functional channel. It was demonstrated that most of this almost thirty-member family transporters are activated by a variety of different stimuli and function as signal integrators. The most examined intracellular TRPs modulators are cytosolic calcium binding proteins and membrane anchored phosphatidylinositol phosphates (PIPs). These signal integrators bind specific domains in intracellular termini of TRPs, thereby change their structure and activate or inhibit the transportation function of receptor. To identify a novel ligand...
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The mechanism of the regulation of phosducin function
Kacířová, Miroslava ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Pavlíček, Jiří (referee)
This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...
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