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.

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 °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 °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–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.

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 transient receptor potential vanilloid 3 (TRPV3) channel is a Ca2+-permeable thermosensitive ion channel widely expressed in keratinocytes, where together with epidermal growth factor receptor (EGFR), it forms a signaling complex regulating epidermal homeostasis. Proper signaling through this complex is achieved and maintained via several pathways in which TRPV3 activation is absolutely required. Results of recent studies have suggested that low-level constitutive activity of TRPV3 induces EGFR-dependent signaling that, in turn, amplifies TRPV3 via activation of the mitogen-activated protein kinase ERK in a positive feedback loop. Here, we explored the molecular mechanism that increases TRPV3 activity through EGFR activation. We used mutagenesis and whole-cell patch-clamp experiments on TRPV3 channels endogenously expressed in an immortalized human keratinocyte cell line (HaCaT) and in transiently transfected HEK293T cells and found that the sensitizing effect of EGFR on TRPV3 is mediated by ERK. We observed that ERK-mediated phosphorylation of TRPV3 alters its responsiveness to repeated chemical stimuli. Among several putative ERK phosphorylation sites, we identified threonine 264 in the N-terminal ankyrin repeat domain as the most critical site for the ERK-dependent modulation of TRPV3 channel activity. Of note, Thr-264 is in close vicinity to a structurally and functionally important TRPV3 region comprising an atypical finger 3 and oxygen-dependent hydroxylation site. In summary, our findings indicate that Thr-264 in TRPV3 is a key ERK phosphorylation site mediating EGFR-induced sensitization of the channel to stimulate signaling pathways involved in regulating skin homeostasis.