Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin

Moparthi, Lavanya; Moparthi, Satish Babu; Wenger, Jérôme; Zygmunt, Peter M.

doi:10.1101/2020.03.19.999276

Cited by 4 publications

(4 citation statements)

References 23 publications

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“…Interestingly, the N-terminal ARD is not only sensitive to modifications of cysteines and lysines but also to single point mutations of other amino acids changing the mTRPA1 temperature properties from cold to heat-sensitive [ 28 ]. It is also important to take into consideration that other known intracellular modifiers of TRPA1 (e.g., pH, Ca 2+ , polyphosphates) as well as temperature, pressure and membrane potential [ 1 , 2 , 27 , 29 , 30 ], alone or in combination, may change channel conformation and the prerequisite for the N-terminal ARD in TRPA1 gating.…”

Section: Discussionmentioning

confidence: 99%

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry

Moparthi

Kjellström

Kjellbom

et al. 2020

IJMS

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The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.

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Section: Discussionmentioning

confidence: 99%

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry

Moparthi

Kjellström

Kjellbom

et al. 2020

IJMS

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“…Likewise, the heat-sensitivity of Ag-TRPA1(A) without its N-ARD (Δ1-776 AgTRPA1) was estimated higher than for Ag-TRPA1(A) for which a Q10 value of 28 could be calculated (8). This suggests a modulatory role of the N-ARD in TRPA1 thermosensation, but may not necessarily be driven by temperature sensitive domains within N-ARD as both calcium responses and mechanosensitivity were different between purified hTRPA1 and Δ1-688 hTRPA1 at 22°C and otherwise identical experimental conditions (23,24). Further deletion of VSLD in hTRPA1 (Δ1-854 hTRPA1), keeping most of the S4-S5 linker with the redox sensitive C856 and N855, the mutation of which in humans causes stress-induced cold hypersensitivity (34,35), created a channel with a dramatic increase in cold sensitivity and lost heat sensitivity, the latter indicating heat sensitive structures within VSLD or its uncoupling from a heat sensitive CTD.…”

Section: Discussionmentioning

confidence: 87%

“…The activity of Δ1-688 hTRPA1 and ∆1-854 hTRPA1 was abolished by the TRPA1 inhibitors HC030031 and ruthenium red (Fig. 1B,C and 2B,C), at concentrations that abolished purified hTRPA1 and Δ1-688 hTRPA1 single-channel activity evoked by chemical ligands, temperature and mechanical stimuli (7,11,(22)(23)(24). Furthermore, no channel activity was observed in membranes, without the purified hTRPA1 proteins in the presence of the detergent Fos-Choline-14, when exposed to the temperatures used in the present study (7,11,(22)(23)(24).…”

Section: Effect Of Temperature On Purified ∆1-688 Htrpa1 and ∆1-854 Htrpa1 Channel Activitymentioning

confidence: 92%

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

Moparthi

Sinica

Filipović

et al. 2021

Preprint

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The human TRPA1 (hTRPA1) is an intrinsic thermosensitive ion channel responding to both cold and heat, depending on the redox environment. Here, we have studied purified hTRPA1 truncated proteins to gain further insight into the temperature gating of hTRPA1. We found in patch-clamp bilayer recordings that Δ1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas Δ1-854 hTRPA1 that is also lacking the S1-S4 voltage sensing-like domain (VSLD) gained sensitivity to cold but lost its heat sensitivity. The thiol reducing agent TCEP abolished the temperature sensitivity of both Δ1-688 hTRPA1 and Δ1-854 hTRPA1. Cold and heat activity of Δ1-688 hTRPA1 and Δ1-854 hTRPA1 were associated with different structural conformational changes as revealed by intrinsic tryptophan fluorescence measurements. Heat evoked major structural rearrangement of the VSLD as well as the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD), whereas cold only caused minor conformational changes. As shown for Δ1-854 hTRPA1, a sudden drop in tryptophan fluorescence occurred within 25-20°C indicating a transition between heat and cold conformations of the CTD, and thus it is proposed that the CTD contains a bidirectional temperature switch priming hTRPA1 for either cold or heat. In whole-cell patch clamp electrophysiology experiments, replacement of the cysteines 865, 1021 and 1025 with alanine modulated the cold sensitivity of hTRPA1 when heterologously expressed in HEK293T cells. It is proposed that the hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

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“…[8][9][10][11][12] The channel is additionally activated by, and may act as a sensor for, acidity (protons) 13 and calcium. [14][15][16] TRPA1 is expressed in nociceptive neurons, 17 where it acts directly in the inception of pain and itch, but has also been found in the vascular endothelium 18 and lung epithelium 19 among others places. Significantly, it has been shown to be activated by endogenous inflammation messengers such as bradykinin 19 and nitrooleic acid 20 (as well as miRNAs 21 ), and hence appears to play a role in inflammation of critical tissues.…”

Section: Trpa1 (Transient Receptor Potential Ankyrin 1 or Transient Receptor Potential Channelmentioning

confidence: 99%

Atomistic Mechanisms of Human TRPA1 Activation By Electrophile Irritants Through Molecular Dynamics Simulation and Mutual Information Analysis

Habgood

Seiferth

Zaki

et al. 2021

Preprint

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The ion channel TRPA1 is a promiscuous chemosensor, with reported response to a wide spectrum of noxious electrophilic irritants, as well as cold, heat, and mechanosensation. It is also implicated in the inception of itch and pain and has hence been investigated as a drug target for novel analgesics. The mechanism of electrophilic activation for TRPA1 is therefore of broad interest. TRPA1 structures with the pore in both open and closed states have recently been published as well as covalent binding modes for electrophile agonists. However, the detailed mechanism of coupling between electrophile binding sites and the pore remains speculative. In addition, while two different cysteine residues (C621 and C665) have been identified as critical for electrophile bonding and activation, the bound geometry has only been resolved at C621. Here, we use molecular dynamics simulations of TRPA1 in both pore-open and pore-closed states to explore the allosteric link between the electrophile binding sites and pore stability. Our simulations reveal that an open pore is structurally stable in the presence of open ‘pockets’ in the C621 / C665 region, but rapidly collapses and closes when these pockets are shut. Binding of electrophiles at either C621 or C665 provides stabilisation of the pore-open state, but molecules bound at C665 are shown to be able to rotate in and out of the pocket, allowing for immediate stabilisation of transient open states. Finally, mutual information analysis of trajectories reveals an informational path linking the electrophile binding site pocket to the pore via the voltage-sensing-like domain, giving a detailed insight into the how the pore is stabilized in the open state.

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Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin

Cited by 4 publications

References 23 publications

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry

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

Atomistic Mechanisms of Human TRPA1 Activation By Electrophile Irritants Through Molecular Dynamics Simulation and Mutual Information Analysis

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