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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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Modulatory mechanisms of nociceptive TRP channels
Maršáková, Lenka ; Vlachová, Viktorie (advisor) ; Novotný, Jiří (referee) ; Zemková, Hana (referee)
Detection of painful stimuli in the periphery is mediated by temperature-sensitive transient receptor potential (TRP) channels which are expressed in primary afferent endings of free sensory neurons called nociceptors. TRP channels in nociceptors are involved in the detection of thermal, but also mechanical and chemical stimuli. Out of seven known types of temperature-sensitive TRP channels, three are responsible for detecting painful temperatures: vanilloid receptors TRPV1 (> 42 o C) and TRPV2 (> 52 o C) detect noxious heat, and ankyrin receptor TRPA1 detects noxious cold (< 17 o C). Better knowledge of TRP channel mechanisms of action is essential for understanding TRP channel functions and ultimately for the design of potential analgesics. New findings presented in this thesis clarify mechanisms of action of TRPV1 and TRPA1 receptors, focusing on camphor and voltage sensitivity of TRPV1 channels and calcium modulation of TRPA1 channels. The first topic discussed in this thesis is the mechanism of camphor sensitivity of TRPV1 receptor. Camphor is a naturally occurring substance known since time immemorial for its effective analgesic properties, yet its mechanism of action is not understood. Camphor is known to be a partial agonist of TRPV1 channel, a full agonist of TRPV3 channel, but also an inhibitor of...
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Struktura a funkce rekombinantního P2X4 receptoru
Rokič, Miloš ; Zemková, Hana (advisor) ; Vlachová, Viktorie (referee) ; Bendová, Zdeňka (referee)
4 Abstract Purinergic P2X receptors are membrane ion channels activated by extracellular ATP. There are seven isoforms of mammalian P2X receptors designated as P2X1-7, which according to their structure represent a specific family of ligand gated ionic channels, with extraordinary structural/functional properties. The P2X receptor consists of three subunits and each subunit has two transmembrane domains. Crystalographic data demonstrate that ionic channel pore is situated between the second transmembrane domains. Crystal structure of P2X4 receptor from the zebrafish (Danio rerio) is available in both open and closed state of the channel and the exact structure of ATP binding site is solved. The aim of this thesis was to study the structure-function relationships in a model of recombinant P2X4 receptor of the rat. By employing the point mutagenesis and electrophysiological recording, the functional importance of conserved cysteine residues in the ectodomain and amino acid residues which form the extracellular vestibule was investigated. All ten cysteins were substituted one by one with alanine or threonine and ATP-induced currents were measured from HEK293T cells expressing wild type (WT) and mutated P2X4 receptors. The results indicate that C116A, C126A, C149A and C165A mutations disrupt two disulfide bonds...
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Mechanisms of activation and modulation of vanilloid TRP channels
Boukalová, Štěpána ; Vlachová, Viktorie (advisor) ; Hock, Miroslav (referee) ; Zemková, Hana (referee)
Štěpána Boukalová Mechanisms of activation and modulation of vanilloid TRP channels TRPV1 and TRPV3 are thermosensitive ion channels from the vanilloid subfamily of TRP receptors. TRPV1, which is primarily expressed in nociceptive sensory neurons, is an important transducer of painful stimuli and is also involved in the detection of noxious heat. TRPV3 is expressed mainly in the skin where it regulates proliferation and differentiation of keratinocytes. Similarly to voltage-dependent potassium (Kv) channels, TRP receptors are comprised of four subunits, each with six transmembrane segments (S1-S6). Using mutational approach, we tried to elucidate the role of S1 in TRPV1 functioning. Our results indicate that the extracellular portion of S1 plays a crucial role in TRPV1 gating. TRPV1 channels with a conservative mutation of positively charged residue in this region (R455K substitution) were overactive. However, they were neither activated nor potentiated by low pH; on the contrary, protons stabilized the closed conformation of this mutant channel. Very similar phenotypic properties were found in other TRPV1 mutants with substitution in S4/S5-S5 region and in the pore helix. In Kv channels, extracelular portion of S1 forms a small contact surface with the pore helix, which allows efficient transmission of...
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Expression and functional characteriz ation of transient receptor potential vanilloid - related channel 4 (TRPV4) in hippocampal astrocytes after ischemia /reperfusion
Butenko, Olena ; Anděrová, Miroslava (advisor) ; Bojar, Martin (referee) ; Vlachová, Viktorie (referee)
The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by cell swelling, low pH and high temperature may be involved in neuronal and glia pathophysiology. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are...
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Thermally gated TRP channels in nociceptive neurones
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Mrózková, Petra (referee)
Transduction ion channels are gated in response to a variety of external stimuli and this process is critical for the proper functioning of sensory neurons. These specialized proteins enable the survival of any organism, which depends on having adequate information about the external environment. The thermosensitive TRP (transient receptor potential) ion channels, whose molecular structure has been identified during last decades, enable the transduction of thermal stimuli in primary nociceptive neurons. During the last decade, molecular biological techniques have provided new tools for studying the structure of these specialized transduction ion channels in relation to their function and to understand more deeply their physiological roles. The aim of this bachelor thesis is to give an overview of recent evidence regarding the functional and physiological properties of sensory-neuron specific mammalian TRP ion channels that are activated by thermal stimuli: heat and cold.
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