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Study of the relations between the structure and function of C - terminal vanilloid receptor TRPV1
Gryčová, Lenka ; Obšil, Tomáš (advisor) ; Heřman, Petr (referee) ; Urbánková, Eva (referee) ; Krůšek, Jan (referee)
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labeling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations. Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the Cterminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT's binding to CaM, with arginine R785...
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The role of TRPV1 receptors in chemokine CCL2 induced modulation of nociceptive synaptic transmission at spinal cord level
Adámek, Pavel ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn is a significant mechanism in the development and maintenance of different pathological pain states. Accumulating evidence indicates that the TRPV1 (transient receptor potential vanilloid 1) receptor and chemokine CCL2 (C-C motif ligand 2) may play a critical role in this process. The aim of this diploma thesis was to investigate the CCL2 induced modulation of nociceptive synaptic transmission in the dorsal horn of spinal cord and the role of the TRPV1 receptors. To investigate this aim patch-clamp recordings of spontaneous and miniature excitatory postsynaptic currents (sEPSC, mEPSC) from superficial dorsal horn neurons in acute rat lumbar spinal cord slices were used. After acute application of CCL2 on the slice preparation from naïve animals, a frequency increase of both sEPSC and mEPSC was present. This CCL2 induced increase in both sEPSC and mEPSC frequency was prevented by the TRPV1 receptor antagonist SB366791 application. No changes were observed in the amplitudes of sEPSC or mEPSC after application of the CCL2, SB366791, or co-application of CCL2 and SB366791. This suggests that the observed changes were mediated predominantly by presynaptic mechanisms. The preliminary results indicate that after chronic constriction...
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Study of the factors affecting the binding specificity of the 14-3-3 proteins.
Veisová, Dana ; Obšilová, Veronika (advisor) ; Bařinka, Cyril (referee) ; Krůšek, Jan (referee)
113 11. Summary The 14-3-3 proteins are dimeric molecules with a characteristic shape and molecular mass about 30 kDa found in all eukaryotes. They are playing a key role in a variety of biological processes such as signal transduction, cell differentiation and apoptosis. The C- terminal segment of human 14-3-3ζ plays an important role as an autoinhibitor which can occupy the ligand binding groove in the absence of binding partner and blocks the binding of inappropriate ligand. The C-terminal segment structure has not been identified for any of the known crystallographic structures. Unlike the helical region α1-α9, the C-terminal segment shows the highest sequence variability. It is believed that the C-terminal segment is the most flexible region and can exist in a lot of conformations. The yeast isoforms of the 14-3-3 proteins Bmh1 and Bmh2 possess a distinctly variant C-terminal segment which is longer and contains a polyglutamine stretch of unknown function. The role of this C-terminal part has been studied with many of different biophysical methods. Dynamic light scattering, sedimentation velocity, time resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that an apparent size of the molecules Bmh1 and Bmh2 is significantly bigger compared to the 14-3-3 isoforms....
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Modulation of synaptic transmission, studies on spinal cord slices in vitro
Mrózková, Petra ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of a synaptic transmission in the spinal cord dorsal horn plays a key role in nociceptive signalling, especially in states of pathological pain. The goal of this study was to develop a method for calcium imaging in spinal cord slices in vitro. This method allowed us to record changes of intracellular free calcium ions concentration (iCa2+ ), that are a major mediator of neuronal plasticity. In this work, we have focused on application of this method in a conventional fluorescence microscope and on the role of different neuromodulators of synaptic activity. Changes of iCa2+ induced by dorsal root electrical stimulation were recorded altogether in 744 dorsal horn (lamina I and II) neurons. In the first series of experiments, stimulation protocols activating preferentially A and A + C dorsal root fibers were used and long-term stability of the calcium responses was verified. The dorsal root stimulation induced in the neurons fast and delayed type of calcium response. Application of AMPA and NMDA receptors antagonists, CNQX (50μM) and MK801 (45μM), reduced the calcium response amplitude and confirmed the importance of glutamate receptors in synaptic activation. In several experiments the effect of capsaicin a TRPV1 receptors agonist, application was tested. Application of even low...
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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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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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Calcium homeostasis and modulation of nociceptive synaptic transmission
Sojka, David ; Paleček, Jiří (advisor) ; Žurmanová, Jitka (referee) ; Krůšek, Jan (referee)
2 SUMMARY OF THE THESIS This study was designed to improve our knowledge regarding mechanisms of nociceptive signaling at spinal cord level. One of the forms of spinal cord synaptic transmission modulation is central sensitization, a manifestation of synaptic plasticity at spinal cord level, which was found to be present at many chronic pain syndromes. This study deals mainly with a development of calcium imaging technique with a final goal to study mechanisms of central sensitization in vitro on population of dorsal horn neurons. We have analyzed synaptically evoked intracellular Ca changes as a result of dorsal root stimulation in a superficial dorsal horn area in spinal cord slices and found two types of Ca responses: one synchronized with electrical stimulation and a second one, delayed response due to Ca release from internal stores. The delayed Ca release was not previously shown to be present in these neurons and it was not dependent on activation of ionotropic glutamatergic receptors, suggesting involvement of metabotropic receptor pathway. The presence of this delayed type of Ca response could have a significant role in the induction of some types of chronic pain syndromes since intracellular calcium increase is thought to be a key trigger point in spinal cord neurons sensitization. An important...
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