|
|
Pathological pain states, the role of synaptic modulation at spinal cord level
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...
|
|
|
Mobilization of intracellular calcium via muscarinic acetylcholine receptors.
Šantrůčková, Eva ; Jakubík, Jan (advisor) ; Krůšek, Jan (referee)
Muscarinic acetylcholine receptors play important role in many physiological and pathophysiological processes. Muscarinic receptors are metabotropic receptors for acetylcholine. The objective of this thesis is to compare long-term effects of short-term exposure of the muscarinic receptors to xanomeline among subtypes. There are two groups of muscarinic receptors that differ in their extracellular-to-intracellular signal transduction - the odd-numbered ones (M1, M3, M5) preferentially couple to Gq/11 G-proteins, whereas even-numbered ones (M2, M4) couple mainly via Gi/o. CHO cells stably expressing individual muscarinic receptor subtypes were used for all experiments. Cells expressing M2 and M4 receptors were transiently transfected with cDNA for human Gq/16 G-protein to achieve calcium response comparable to the one at oddnumbered subtypes. Calcium level was measured with the fluorescent indicator Fura 2. Xanomeline stimulation induced the fast mobilization of the intracellular calcium at all five receptor subtypes. In accordance with the functional selectivity of xanomeline for M1 and M4 receptors, the immediate calcium response to xanomeline was comparable to that of full agonist carbachol and the elevated calcium level sustained for one hour after xanomeline had been removed. On the other hand,...
|
|
| |
|
| |
|
| |
|
|
Měření membránového potenciálu u mutantních kmenů Saccharomyces cerevisiae deficientních v různých membránových transportérech
Džamba, Dávid ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
Potassium transport across the cell membrane is supposed to contribute to the maintenance of membrane potential. In this work, the diS-C3(3) assay was used to determine the contribution of three main potassium transporters of S. cerevisiae cells (Trk1p, Trk2p and Tok1p) to the membrane potential changes in three different growth phases. It was shown that deletions of these transporters cause hyperpolarisation of the cell membrane in exponential and early diauxic growth phases; no difference was detected in post-diauxic cells. Another contribution of this work is in a deeper study of the activity of ABC pumps which are crucial in multidrug resistance. It was proved that apart from Pdr5p and Snq2p pumps, there are also other active extrusion pumps (most likely Pdr10p and Pdr15p). It was also showed that pdr1, pdr3 mutant strain is not an equivalent of strain lacking the multidrug pumps Pdr5p, Snq2p and Yor1p. The results in this work indicate that dis-C3(3) probe is probably a substrate of other pumps such as Pdr15p. Study of these transporters with our method is thus very suitable and could provide detailed information about their kinetics and help in finding their inhibitors.
|
|
|
Study of factors influencing the function of MntH, membrane transport protein of E. coli
Jurková, Ingrid ; Urbánková, Eva (advisor) ; Krůšek, Jan (referee)
MntH belongs to the Nramp family of transport proteins, and plays an important role not only in homeostasis of iron and manganese, but also in bacterial defence against the immunity response of an infected host cell. MntH co-transports divalent metal ions into the cell together with protons with a stoichiometry dependent on the membrane potential and extracellular pH. Using the redistribution potential dye diS-C3(3), we measured the effects of MNTH expression and MntH-mediated metal transport on the cell membrane potential and intracellular pH. Cells expressing MNTH were found to be hyperpolarised and their membrane potential was depolarised upon the addition of metal ions. In the theoretical part of our work, we explored general four-, six-, and eight-state carrier models that were modified by introducing the voltage dependence of all rate constants. Using mathematical modelling, we simulated the effect of various model parameters (including membrane potential, substrate concentration, and carrier or substrate charge) on substrate influx. We observed some of the transport characteristics described for MntH proteins such as variable symport stoichiometry that is influenced by the membrane potential and pH. However, for a more detailed simulation of the eight-state carrier model, more information about...
|
|
|
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...
|
|
|
Topology and function of the transmembrane domain of colicin U produced by Shigella boydii
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee)
Colicin U is a protein produced by strains of bacterium Shigella boydii. It exhibits antibacterial activity against some bacterial strains Shigella and Escherichia. Based on sequence homology with colicins A, B and N, the colicin U is classified as a pore-forming colicin. Interaction of colicin U with attacked bacteria is ensured by three-step mechanism: 1) First colicin U interacts with surface receptors OmpA, OmpF and core of LPS. 2) Thereafter the colicin is translocated to periplasm through interaction with Tol proteins. 3) Finally colicin U interacts with the inner membrane of the attacked bacteria causing its depolarization. In this thesis I demonstrated pore-forming features of colicin U and further observed characteristics and properties of these pores. Using methods of measuring on black lipid membranes I determined a single channel conductance (19 pS), ion selectivity, the influence of various conditions on the behaviour of the pores. These findings, in many cases, correspond to the findings on other related colicins. Furthermore, I successfully determined the pore diameter of colicin U ( ≈ 0,8 nm). The next section of the thesis focuses on creation of single cysteine mutations of colicin U. Subsequently I produced five mutant variants of colicin U and verified their functionality so that...
|
|
| |
guest ::
