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Functional role of cytoplasmic domains in the gating of TRPA1 channel
Vašková, Jana ; Vlachová, Viktorie (advisor) ; Zemková, Hana (referee)
The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in a subset of primary afferent neurones where it is activated by a variety of pungent and chemically reactive compounds such as allyl isothiocyanate or cinnamaldehyde. This voltage- dependent channel is activated through covalent modification of cytoplasmic cysteines and, from the cytoplasmic side, is also critically regulated by calcium ions. Both, amino (N-) and carboxyl (C-) termini have been shown to be involved in these processes. Using electrophysiological and molecular-biology techniques, we explored the role of specific cytoplasmic domains in the activation of TRPA1. By measuring chemically-, voltage-, and calcium-activated membrane TRPA1-mediated currents, we identified highly conserved serine and threonine residues along the N-terminal ankyrin repeat domain, mutation of which strongly affected responses of the channel. In addition, using C-terminally truncated construct previously reported to be involved in calcium regulation, we present a new finding that the distal C-terminal tail contributes to voltage-dependent activation of TRPA1.
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Molecular mechanisms of activation and modulation of TRPV3 receptor
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Novotný, Jiří (referee)
Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...
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The mechanism of regulation of NMDA receptors transport to the cell surface
Lichnerová, Katarína ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are a class of ionotropic glutamate receptors, involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They form heterotetrameric complexes composed of GluN1, GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Previous reports showed that different subunits of NMDA receptors, especially the GluN2 subunits, confer different functional and pharmacological properties on the receptor complexes. However, the subunit-dependent differences in the regulation of intracellular processing and transport of NMDA receptor subtypes has not been clearly elucidated. The aim of this work was to clarify the mechanisms of regulation of the NMDA receptor transport. In our experiments we performed immunocytochemistry of receptors on heterologous COS-7 cells and cultured cerebellar granule cells (CGC), both expressing recombinant NMDA receptors. The results of my work show that the transport of NMDA receptors is regulated by presence of GluN2A and GluN2B subunits. Our results further showed that transport of the GluN1/GluN2C receptors is regulated by three specific areas of the GluN2C subunit: i) the A2 segment within the amino- terminal domain, ii.) the M3 domain, and iii.) the proximal part of the C-terminus containing the...
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Functional and structural study of thermally activated TRP ion channels: The role evolutionarily conserved motifs in the TRPA1 modulation
Kádková, Anna
Ankyrin receptor TRPA1 is an ion channel widely expressed on primary afferent sensory neurons, where it acts as a polymodal sensor of nociceptive stimuli. Apart from pungent chemicals (e. g. isothiocyanates, cinnamaldehyde and its derivatives, acrolein, menthol), it could be activated by cold temperatures, depolarizing voltages or intracellular calcium ions. TRPA1 channel is a homotetramer in which each subunit consists of cytoplasmic N and C termini and a transmembrane region. The transmembrane part is organized into six alpha- helices connected by intra- and extracellular loops. The N terminus comprises a tandem set of 16 to 17 ankyrin repeats (AR), while the C terminus has a substantially shorter, dominantly helical structure. In 2015, a partial cryo-EM structure of TRPA1 was resolved; however, the functional roles of the individual regions of the receptor have not yet been fully understood. This doctoral thesis is concerned to elucidate the role of highly conserved sequence and structural motifs within the cytoplasmic termini and the S4-S5 region of TRPA1 in voltage- and chemical sensitivity of the receptor. The probable binding site for calcium ions that are the most important physiological modulators of TRPA1 was described by using homology modeling, molecular-dynamics simulations,...
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Photocontrolled Biomolecules
Planer, Jakub ; Bartošík, Miroslav (referee) ; Vácha,, Robert (referee) ; Kulhánek, Petr (advisor)
This work is focused on molecular dynamics simulations of artificial photosensitive ion channel and AFM probe. To assemble this ion channel, DFT methods were employed for reparametrization of the GAFF force field describing the bridged azobenzene, which was used as a light controlled molecular switch. We proved by molecular dynamics simulations that newly developed parameters correctly describe behavior of assembled model of ion channel in a lipid bilayer. We also constructed a model of AFM probe and observed formation of water meniscus between the AFM probe and surface, both made of -quartz, by employing molecular dynamics simulations. A contribution of this work is the set of new parameters extending GAFF force field for description of the bridged azobenzene. We also verified functionality of ion channel model and model of AFM probe, which can be used for the further water meniscus studies.
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