Molekulární mechanismy polymodální regulace TRPA1 receptoru

The TRPA1 channel is a universal, nociception-mediating cellular sensor activated by various environmental irritants, potentially harmful physical modalities and endogenous mediators of pathophysiological processes. The polymodality of TRPA1 channel allows the activation stimuli to further enhance or suppress each other's effect. While this modulation effect has its physiological importance in promoting the protective cellular and behavioral mechanisms, it may result into the unpleasant pain-related effects accompanying the chronical pain caused by aberrant TRPA1 channel activity. In order to effectively and selectively target the synergic properties of TRPA1 modulators, while preserving the sensitivity to the environmental threads, the knowledge of the mechanisms of polymodal regulation at the molecular level are required. This doctoral thesis aims at the elucidation of three main mechanisms of TRPA1 regulation: 1) the regulation via intracellular signaling cascades and phosphorylation, 2) the interaction with membrane phospholipids and 3) the temperature-driven gating. The results presented in the thesis show that the effects of the inflammatory mediator bradykinin are decreased by the low-frequency high-induction electromagnetic field used in magnetotherapy. We have identified a residue S602...TRPA1 kanál je univerzální buněčný nociceptivní senzor, který je aktivován celou řadou vnějších podnětů potenciálně škodlivých modalit a různých endogenních mediátorů produkovanými v důsledku patofyziologických procesů. Polymodalita TRPA1 kanálu umožňuje zesílit či zeslabit účinek aktivačního podnětu přítomností dalšího stimulu. Tento modulační efekt je zásadní pro aktivizaci ochranných buněčných a behaviorálních mechanismů, může však také vést k nežádoucím účinkům v případě chronických patofyziologických stavů způsobených nadměrnou aktivací TRPA1 kanálu. Aby bylo možné účinně a selektivně zacílit mechanismy synergických účinků TRPA1 aktivátorů a zároveň zachovat fyziologickou úlohu iontového kanálu, je nezbytné poznat mechanismy polymodální regulace TRPA1 na molekulární úrovni. Předložená disertační práce se zabývá třemi důležitými mechanismy regulace TRPA1 kanálu: 1) regulací buněčnými signálními kaskádami a fosforylací, 2) interakcí s membránovými fosfolipidy a 3) regulací aktivity změnami okolní teploty. Výsledky uvedené v disertační práci ukazují, že účinek mediátoru zánětu bradykininu je snížen stimulací nízkofrekvenčním vysoko-indukčním elektromagnetickým polem užívaným při magnetoterapii. Identifikovali jsme serin 602, jehož fosforylace může potenciálně inhibovat aktivitu TRPA1 kanálu. Dále...Department of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, ∆1–688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1–854 hTRPA1, also lacking the S1–S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5–S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5–S6 pore region and the VSLD, respectively

Molecular mechanisms of polymodal regulation of TRPA1 receptor

The TRPA1 channel is a universal, nociception-mediating cellular sensor activated by various environmental irritants, potentially harmful physical modalities and endogenous mediators of pathophysiological processes. The polymodality of TRPA1 channel allows the activation stimuli to further enhance or suppress each other's effect. While this modulation effect has its physiological importance in promoting the protective cellular and behavioral mechanisms, it may result into the unpleasant pain-related effects accompanying the chronical pain caused by aberrant TRPA1 channel activity. In order to effectively and selectively target the synergic properties of TRPA1 modulators, while preserving the sensitivity to the environmental threads, the knowledge of the mechanisms of polymodal regulation at the molecular level are required. This doctoral thesis aims at the elucidation of three main mechanisms of TRPA1 regulation: 1) the regulation via intracellular signaling cascades and phosphorylation, 2) the interaction with membrane phospholipids and 3) the temperature-driven gating. The results presented in the thesis show that the effects of the inflammatory mediator bradykinin are decreased by the low-frequency high-induction electromagnetic field used in magnetotherapy. We have identified a residue S602..

Human and Mouse TRPA1 Are Heat and Cold Sensors Differentially Tuned by Voltage

Transient receptor potential ankyrin 1 channel (TRPA1) serves as a key sensor for reactive electrophilic compounds across all species. Its sensitivity to temperature, however, differs among species, a variability that has been attributed to an evolutionary divergence. Mouse TRPA1 was implicated in noxious cold detection but was later also identified as one of the prime noxious heat sensors. Moreover, human TRPA1, originally considered to be temperature-insensitive, turned out to act as an intrinsic bidirectional thermosensor that is capable of sensing both cold and heat. Using electrophysiology and modeling, we compare the properties of human and mouse TRPA1, and we demonstrate that both orthologues are activated by heat, and their kinetically distinct components of voltage-dependent gating are differentially modulated by heat and cold. Furthermore, we show that both orthologues can be strongly activated by cold after the concurrent application of voltage and heat. We propose an allosteric mechanism that could account for the variability in TRPA1 temperature responsiveness

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The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2 ) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP2 -interaction site (Y1006-Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992-N1008 and T1003-P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. Using whole-cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage-dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide-interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate

The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, Delta 1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas Delta 1-854 hTRPA1, also lacking the S1-S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.Funding Agencies|Swedish Research Council [2014-3801]; Medical Faculty of Lund University-ALF [ALFSKANE451751]; Hjarnfonden/the Swedish Brain Foundation [FO20200188]; Stiftelsen Olle Engkvist Byggmastare [189-290]; Albert Pahlssons stiftelse; Alfred Osterlunds stiftelse; Czech Science Foundation [22-13750S]; European Unions Horizon 2020 research and innovation programunder [101004806]</p

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials

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Odontoblast TRPC5 channels signal cold pain in teeth.

peer reviewedTeeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system