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The role of nociceptive synaptic transmission modulation at the spinal cord level in different pain states
Adámek, Pavel ; Paleček, Jiří (advisor) ; Vaculín, Šimon (referee) ; Vlachová, Viktorie (referee)
Pain is a common symptom of many clinical syndromes and diseases. In particular, the treatment of neuropathic pain represents a serious public health issue because currently available analgesia is ineffective in many cases or it has adverse effects. Treatment of pain-related suffering requires knowledge of how pain signals are initially generated and subsequently transmitted by the nervous system. A nociceptive system plays a key role in this process of encoding and transmission of pain signals. Modulation of the nociceptive synaptic transmission in the spinal cord dorsal horn represents an important mechanism in the development and maintenance of different pathological pain states. This doctoral thesis has aimed to investigate and clarify some of the mechanisms involved in the modulation of the spinal nociceptive processing in different pain states. The main attention was paid to study the following issues: (I.) Which is the role of Transient Receptor Potential Vanilloid type 1 channels (TRPV1), Toll-Like Receptors 4 (TLR4), and phosphatidylinositol 3-kinase (PI3K) in the development of neuropathic pain induced by paclitaxel (PAC) chemotherapy in acute in vitro, and subchronic in vivo murine model of PAC-induced peripheral neuropathy (PIPN)? (II.) How is affected spinal inhibitory synaptic control...
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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 ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee) ; Obšilová, Veronika (referee)
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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The role of charged residues in the activation and modulation of the TRPA1 ion channel
Zímová, Lucie ; Vlachová, Viktorie (advisor) ; Doležal, Vladimír (referee) ; Rokyta, Richard (referee)
Important receptor for sensing painful stimuli is ion channel TRPA1, which is expressed in peripheral endings of nociceptive neurons, where it serves as transducer of physical and chemical environmental signals to the language of the nervous system. The effort to understand the mechanisms of its activity on a molecular level is driven by the vision of progress in treatment of chronic pain in humans. Our work focused on C-terminal cytoplasmic domain of TRPA1 receptor, where we described i.a. the probable binding site for calcium, which is the most important TRPA1 modulator. Using the combination of homology modeling and molecular dynamic simulations with electrophysiological measurements we were able to explain molecular basis of familial pain syndrome caused by TRPA1 point mutation. We contributed to the understanding of the TRPA1 voltage-dependent activation mechanism by describing the amino acids in proximal C-terminus and in S4-S5 linker of transmembrane domain that are directly involved in voltage-dependent gating. Powered by TCPDF (www.tcpdf.org)
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Role of reactive cysteines in the activation of the human TRPA1 ion channel
Synytsya, Viktor ; Vlachová, Viktorie (advisor) ; Zemková, Hana (referee)
TRPA1 is a thermosensitive ion channel from the family of TRP (transient receptor potential) receptors. In primary sensory neurons, TRPA1 is an important transducer of painful stimuli, where it contributes to detection of noxious, irritant and inflammatory compounds of endogenous and exogenous origin. The major activation mode of TRPA1 is covalent modification of N-terminal cysteines or lysines by electrophilic compounds. The potency of the electrophilic agonists is increased by voltage dependency of the TRPA1 channel, which contributes substantially during membrane depolarization. To date, the role of several cysteine residues in the N- terminus has been demonstrated. However, the functional role of six cysteines in the transmembrane domain is still unknown. The first part of the thesis focuses on the functional role of the transmembrane cysteines in the activation of human TRPA1 channel. Our results indicate that these sites do not mediate reactive-electrophile-induced activation but four of the six cysteines substantially contribute to voltage-dependent gating of the channel and two participate in calcium-dependent modulation of TRPA1. In the second part of this thesis we aim to explore the proximity of two specific charged residues, located in the linker between the fourth and the fifth...
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Mechanisms of signal transduction via the muscarinic receptors
Dolejší, Eva ; Doležal, Vladimír (advisor) ; Kršiak, Miloslav (referee) ; Vlachová, Viktorie (referee)
Muscarinic acetylcholine receptors (mAChR) belong to the family of G-protein coupled receptors. There are five subtypes of mAChR denoted M1 to M5 that are widely and differentially distributed in both the central nervous system and periphery and play an important role in many specific physiological functions. Impairment of muscarinic neurotransmission occurs in serious disorders such as Alzheimer's disease, schizophrenia or Parkinson's disease that are accompanied by cognitive decline mainly due to the disruption of M1 receptor signaling in the brain. Unfortunately, the high degree homology of the orthosteric binding site among muscarinic receptor subtypes makes it very difficult to obtain subtype- selective agonists. One of the few known selective agonists is xanomeline that preferentially activates the M1 and M4 subtypes. Xanomeline exerts unique interactions with muscarinic receptors comprising reversible binding to the orthosteric domain, and wash-resistant allosteric interaction with a secondary binding site. The basis of xanomeline functional selectivity remains largely unknown. In an attempt to probe into such mechanisms we investigated the immediate and long-term effects of xanomeline on activation of muscarinic receptors, using intact Chinese hamster ovary (CHO) cells expressing individual...
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Role of variable chains at the interface between subunits in forming ATP-binding pocket and function of P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Novotný, Jiří (referee) ; Vlachová, Viktorie (referee)
7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...
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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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