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A study of the temperature sensitivity of the human TRPA1 channel
Barvíková, Kristýna ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Transient receptor potential (TRP) ion channels play important physiological roles in the detection of environmental stimuli that occur primarily at the peripheral terminals of specialized sensory neurons. The recently resolved cryo-electron microscopy structures and molecular biological techniques have provided new tools that enable to study these channels in relation to their function, and thus to understand more deeply their pharmacology and physiology. The aim of this bachelor thesis is to give an overview of the current status of research on the ankyrin TRP channel subtype 1 (TRPA1), a channel activated by diverse irritant chemical stimuli but also by temperature changes. The experimental part is focused on the elucidation of the role of the sensor domain in thermal sensitivity of the TRPA1 channel. Using whole-cell patch-clamp electrophysiological technique, the presented results demonstrate that the sensor is an important determinant of voltage-dependent gating. Mutation of the conserved tyrosine in the center of the sensor resulted in channels with clearly different activation kinetics and increased chemical responses upon increasing the temperature from 25 řC to 35 řC. Key words: TRP ion channel, ankyrin receptor, nociception, structure-function, carvacrol
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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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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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A study of the temperature sensitivity of the human TRPA1 channel
Barvíková, Kristýna ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Transient receptor potential (TRP) ion channels play important physiological roles in the detection of environmental stimuli that occur primarily at the peripheral terminals of specialized sensory neurons. The recently resolved cryo-electron microscopy structures and molecular biological techniques have provided new tools that enable to study these channels in relation to their function, and thus to understand more deeply their pharmacology and physiology. The aim of this bachelor thesis is to give an overview of the current status of research on the ankyrin TRP channel subtype 1 (TRPA1), a channel activated by diverse irritant chemical stimuli but also by temperature changes. The experimental part is focused on the elucidation of the role of the sensor domain in thermal sensitivity of the TRPA1 channel. Using whole-cell patch-clamp electrophysiological technique, the presented results demonstrate that the sensor is an important determinant of voltage-dependent gating. Mutation of the conserved tyrosine in the center of the sensor resulted in channels with clearly different activation kinetics and increased chemical responses upon increasing the temperature from 25 řC to 35 řC. Key words: TRP ion channel, ankyrin receptor, nociception, structure-function, carvacrol
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