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

Amplified Cold Transduction in Native Nociceptors by M-Channel Inhibition

Topically applied camphor elicits a sensation of cool, but nothing is known about how it affects cold temperature sensing. We found that camphor sensitizes a subpopulation of menthol-sensitive native cutaneous nociceptors in the mouse to cold, but desensitizes and partially blocks heterologously expressed TRPM8(transient receptor potential cation channel subfamily M member 8). In contrast, camphor reduces potassium outward currents in cultured sensory neurons and, in cold nociceptors, the cold-sensitizing effects of camphor and menthol are additive. Using a membrane potential dye-based screening assay and heterologously expressed potassium channels, we found that the effects of camphor are mediated by inhibition of K(v)7.2/3 channels subtypes that generate the M-current in neurons. In line with this finding, the specific M-current blocker XE991 reproduced the cold-sensitizing effect of camphor in nociceptors. However, the M-channel blocking effects of XE991 and camphor are not sufficient to initiate cold transduction but require a cold-activated inward current generated by TRPM8. The cold-sensitizing effects of XE991 and camphor are largest in high-threshold cold nociceptors. Low-threshold corneal cold thermoreceptors that express high levels of TRPM8 and lack potassium channels are not affected by camphor. We also found that menthol-like camphor-potently inhibits K(v)7.2/3 channels. The apparent functional synergism arising from TRPM8 activation and M-current block can improve the effectiveness of topical coolants and cooling lotions, and may also enhance TRPM8-mediated analgesia

The C-terminal basic residues contribute to the chemical- and voltage-dependent activation of TRPA1

The ankyrin transient receptor potential channel TRPA1 is a non-selective cationic channel that is expressed by sensory neurons, where it can be activated by pungent chemicals, such as AITC (allyl isothiocyanate), cinnamon or allicin, by deep cooling (<18 °C) or highly depolarizing voltages (>+100 mV). From the cytoplasmic side, this channel can be regulated by negatively charged ligands such as phosphoinositides or inorganic polyphosphates, most likely through an interaction with as yet unidentified positively charged domain(s). In the present study, we mutated 27 basic residues along the C-terminal tail of TRPA1, trying to explore their role in AITC- and voltage-dependent gating. In the proximal part of the C-terminus, the function-affecting mutations were at Lys969, Arg975, Lys988 and Lys989. A second significant region was found in the predicted helix, centred around Lys1048 and Lys1052, in which single alanine mutations completely abolished AITC- and voltage-dependent activation. In the distal portion of the C-terminus, the charge neutralizations K1092A and R1099A reduced the AITC sensitivity, and, in the latter mutant, increased the voltage-induced steady-state responses. Taken together, our findings identify basic residues in the C-terminus that are strongly involved in TRPA1 voltage and chemical sensitivity, and some of them may represent possible interaction sites for negatively charged molecules that are generally considered to modulate TRPA1

Cytoplasmic Inter-Subunit Interface Controls Use-Dependence of Thermal Activation of TRPV3 Channel

The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 &deg;C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 &deg;C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251&ndash;E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3

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

S4-S5 linker is involved in voltage-dependent gating of human transient receptor potential ankyrin 1 channel

The transient receptor potential ankyrin 1 channel (TRPA1) is a versatile sensory channel that is gated by depolarizing voltages, deep cooling, membrane deformation, and structurally diverse compounds which include proalgesic agents such as allyl isothiocyanate. How these disparate stimuli converge on the channel protein to open the ion-conducting pore has not yet been fully resolved. The overall architecture of TRP channels shows clear similarities to that seen in the well characterized voltage-gated potassium channels. Here, activation of the voltage sensors in the fourth transmembrane segment (S4) is transduced into pore opening via coupling of the S4-S5 linker to the C-terminal S6 segment. In TRPA1, the gain-of-function mutation N855S located in the S4-S5 region has been associated with familial episodic pain syndrome. In an attempt to elucidate the role of the S4-S5 linker and its putative interaction(s) with S6 or the first C-terminal helix in the voltage-dependent gating of TRPA1, we used site-directed mutagenesis, whole-cell electrophysiology, single-channel recording, and molecular dynamics simulations. The charge-reversal mutations K868E and K969E resulted in a decrease in the rectification index compared to wild-type TRPA1 channels, and a virtually voltage-independent conductance-voltage (G-V) relationship. This effect was also observed in the adjacent charge-neutralizing mutant H970A, but was less pronounced in charge- reversal H970D. These results indicate that positively charged residues in the S4-S5 linker and the helix adjacent to the C-terminal S6 segment play a vital role in the voltage-dependent gating of TRPA1

Comprehensive thermal preference phenotyping in mice using a novel automated circular gradient assay

Currently available behavioral assays to quantify normal cold sensitivity, cold hypersensitivity and cold hyperalgesia in mice have betimes created conflicting results in the literature. Some only capture a limited spectrum of thermal experiences, others are prone to experimenter bias or are not sensitive enough to detect the contribution of ion channels to cold sensing because in mice smaller alterations in cold nociception do not manifest as frank behavioral changes. To overcome current limitations we have designed a novel device that is automated, provides a high degree of freedom, i.e. thermal choice, and eliminates experimenter bias. The device represents a thermal gradient assay designed as a circular running track. It allows discerning exploratory behavior from thermal selection behavior and provides increased accuracy by providing measured values in duplicate and by removing edge artifacts. Our custom-designed automated offline analysis by a blob detection algorithm is devoid of movement artifacts, removes light reflection artifacts and provides an internal quality control parameter which we validated. The assay delivers discrete information on a large range of parameters extracted from the occupancy of thermally defined zones such as preference temperature and skew of the distribution. We demonstrate that the assay allows increasingly accurate phenotyping of thermal sensitivity in transgenic mice by disclosing yet unrecognized details on the phenotypes of TRPM8-, TRPA1- and TRPM8/A1-deficient mice

Structural modeling and patch-clamp analysis of pain-related mutation TRPA1-N855S reveal inter-subunit salt bridges stabilizing the channel open state

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