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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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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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