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The impact of post-translational modifications on TRPC5 ion channel activation and modulation
Mitro, Michal ; Zímová, Lucie (advisor) ; Dolejší, Eva (referee)
Transient Receptor Potential Canonical 5 (TRPC5), a calcium-permeable ion channel, acts as a versatile receptor in sensory neurons, kidneys, and the brain, impacting inflammatory responses and various types of pain. While post-translational modifications influence TRPC5 gating and membrane trafficking, only a few have been described so far. The identification of phosphorylation sites was based on available high-throughput bioinformatics and mass spectrometry data. Subsequently, functional characterization of these sites was conducted by introducing phospho-mimicking aspartate or phospho-null alanine mutations using site-directed mutagenesis. Utilizing patch-clamp in whole-cell configuration, membrane currents evoked by voltage or agonist stimuli were examined. The results revealed that individual substitutions at the N-terminal S193 and S195 with aspartates significantly slowed the gating kinetics. Additionally, a gain-of-function phenotype was observed with S193A. Molecular dynamics simulations provided insight into how phosphorylation at S193 induces changes in interactions among neighboring subunits. Moreover, biotinylation experiments indicated that the alterations in the activity of the S193 mutations are not due to increased targeting of the channels to the plasma membrane. Taken together,...
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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 TRP ion channels in rheumatoid arthritis pain
Havrilčáková, Gabriela ; Zímová, Lucie (advisor) ; Heleš, Mário (referee)
Rheumatoid arthritis is a disease that affects many people across the world. It is a chronic autoimune disease causing inflammation in joints. The inflammation affects joint lining and causes gradual damage to the joint cartilage. Main symptoms are swelling and pain, The perception of pain is closely related to a group of TRP receptors expressed on sensory neurons. The state of inflammation in the affected joints also has a significant influence on the pain accopanying rheumatoid arthritis, in which participate the cells of the joint lining - synovial fibroblasts. They express a subset of TRP receptors that play a key role in the pathophysiology of arthritis development as well. In relation to pain and inflammation, the most studied TRP receptors are TRPV1 and TRPA1. This bachelor thesis describes the findings on these two receptors in relation to pain in rheumatoid arthritis. However, it also describes the TRPC5 receptor, whose inhibiton has only recently been shown in a mouse model to leads to exacerbation of symptoms. The results discussed in this bachelor's thesis show, that pharmacological modulation of the activity of TRP receptors is a promising way of possible future treatment of rheumatoid arthritis, including its main symptom - pain. Key words: rheumatoid arthritis, transient receptor...
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Cellular mechanisms of TRPA1 channel regulation
Barvíková, Kristýna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee)
TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...
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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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Cellular mechanisms of TRPA1 channel regulation
Barvíková, Kristýna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee)
TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...
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Mechanisms of Activation and Modulation of Ion Channels Specific for Nociceptive Neurones
Touška, Filip ; Vlachová, Viktorie (advisor) ; Paleček, Jiří (referee) ; Tureček, Rostislav (referee)
Human body detects potentially damaging stimuli by specialized sensory nerve endings in the skin, the nociceptors. Their membranes are equipped with ion channels, molecular sensors, coding the outside stimuli into the trains of action potentials and conducting them to the higher brain centers. The most prominent group of transduction ion channels is the transient receptor potential (TRP) channel family followed by ion channels responsible for generation and conduction of action potentials from the periphery to the brain, the voltage-gated sodium channels (VGSCs). Understanding the mechanisms how particular stimulus is encoded and processed is of particular importance to find therapeutics for various types of pain conditions. We characterized the properties of VGSC subtypes NaV1.9 and NaV1.8 at high temperatures. We showed that NaV1.9 undergo large increase in current with increasing temperatures and significantly contribute to the action potential generation in dorsal root ganglion (DRG) neurons. Ciguatoxins (CTXs) are sodium channels activator toxins causing ciguatera fish poisoning, a disease manifested by sensory and neurological disturbances. We elucidated the mechanism of CTX- induced cold allodynia, a pathological phenomenon where normally innocuous cool temperatures are perceived as pain. We...
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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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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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