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) 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, Thr264 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 Thr264 in TRPV3 is a key ERK phosphorylation site mediating EGFR-induced sensitization of the channel to stimulate signaling pathways involved in regulating skin homeostasis.

• Keywords
• TRP channels, ankyrin repeat domain, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), keratinocyte, phosphorylation, transient receptor potential channels (TRP channels),
• MeSH
• Cymenes MeSH
• Epidermal Growth Factor metabolism   MeSH
• ErbB Receptors agonists   metabolism   MeSH
• Phosphorylation drug effects   MeSH
• HEK293 Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• TRPV Cation Channels agonists   chemistry   genetics   metabolism   MeSH
• Keratinocytes drug effects   enzymology   metabolism   MeSH
• Humans MeSH
• MAP Kinase Signaling System * drug effects   MeSH
• Patch-Clamp Techniques MeSH
• Mitogen-Activated Protein Kinase 3 chemistry   genetics   metabolism   MeSH
• Membrane Transport Modulators pharmacology   MeSH
• Monoterpenes pharmacology   MeSH
• Mutation MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Processing, Post-Translational drug effects   MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Boron Compounds pharmacology   MeSH
• Threonine metabolism   MeSH
• Cell Line, Transformed MeSH
• Up-Regulation * drug effects   MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-aminoethoxydiphenyl borate MeSH Browser
• carvacrol MeSH Browser
• Cymenes MeSH
• EGFR protein, human MeSH Browser
• Epidermal Growth Factor MeSH
• ErbB Receptors MeSH
• TRPV Cation Channels MeSH
• Mitogen-Activated Protein Kinase 3 MeSH
• Membrane Transport Modulators MeSH
• Monoterpenes MeSH
• Recombinant Proteins MeSH
• Boron Compounds MeSH
• Threonine MeSH
• TRPV3 protein, human MeSH Browser
• Green Fluorescent Proteins MeSH

Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel involved in pain, inflammation and itching. This channel gates in response to many irritant and proalgesic agents, and can be modulated by calcium and depolarizing voltage. While the closed-state structure of TRPA1 has been recently resolved, also having its open state is essential for understanding how this channel works. Here we use molecular dynamics simulations combined with electrophysiological measurements and systematic mutagenesis to predict and explore the conformational changes coupled to the expansion of the presumptive channel's lower gate. We show that, upon opening, the upper part of the sensor module approaches the pore domain of an adjacent subunit and the conformational dynamics of the first extracellular flexible loop may govern the voltage-dependence of multimodal gating, thereby serving to stabilize the open state of the channel. These results are generally important in understanding the structure and function of TRPA1 and offer new insights into the gating mechanism of TRPA1 and related channels.

• Keywords
• S1–S2 linker, TRP channel, allyl isothiocyanate, ankyrin receptor subtype 1, sensor module, transient receptor potential,
• Publication type
• Journal Article MeSH

Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

• MeSH
• Allosteric Regulation MeSH
• Ankyrin Repeat MeSH
• Cyclin-Dependent Kinase 5 genetics   metabolism   MeSH
• Ion Channel Gating drug effects   genetics   MeSH
• HEK293 Cells MeSH
• TRPA1 Cation Channel genetics   metabolism   MeSH
• Humans MeSH
• Mutation MeSH
• Oligopeptides chemistry   pharmacology   MeSH
• Protein Domains MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK5 protein, human MeSH Browser
• Cyclin-Dependent Kinase 5 MeSH
• TRPA1 Cation Channel MeSH
• Oligopeptides MeSH
• TRPA1 protein, human MeSH Browser