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.

• MeSH
• cytoplazma metabolismus   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPV chemie   genetika   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• simulace molekulární dynamiky MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• epidermální růstový faktor metabolismus   MeSH
• erbB receptory agonisté   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kationtové kanály TRPV agonisté   chemie   genetika   metabolismus   MeSH
• keratinocyty účinky léků   enzymologie   metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém * účinky léků   MeSH
• metoda terčíkového zámku MeSH
• mitogenem aktivovaná proteinkinasa 3 chemie   genetika   metabolismus   MeSH
• modulátory membránového transportu farmakologie   MeSH
• monoterpeny farmakologie   MeSH
• mutace MeSH
• mutageneze cílená MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sloučeniny boru farmakologie   MeSH
• threonin metabolismus   MeSH
• transformované buněčné linie MeSH
• upregulace * účinky léků   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Numerous physiological functions rely on distinguishing temperature through temperature-sensitive transient receptor potential channels (thermo-TRPs). Although the function of thermo-TRPs has been studied extensively, structural determination of their heat- and cold-activated states has remained a challenge. Here, we present cryo-EM structures of the nanodisc-reconstituted wild-type mouse TRPV3 in three distinct conformations: closed, heat-activated sensitized and open states. The heat-induced transformations of TRPV3 are accompanied by changes in the secondary structure of the S2-S3 linker and the N and C termini and represent a conformational wave that links these parts of the protein to a lipid occupying the vanilloid binding site. State-dependent differences in the behavior of bound lipids suggest their active role in thermo-TRP temperature-dependent gating. Our structural data, supported by physiological recordings and molecular dynamics simulations, provide an insight for understanding the molecular mechanism of temperature sensing.

• MeSH
• buněčné linie MeSH
• elektronová kryomikroskopie MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPV metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• lipidy chemie   MeSH
• myši MeSH
• nízká teplota MeSH
• termodynamika MeSH
• vazba proteinů fyziologie   MeSH
• vnímání teploty fyziologie   MeSH
• vysoká teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.

• MeSH
• elektrofyziologie metody   MeSH
• iontové kanály chemie   MeSH
• kationtové kanály TRP metabolismus   MeSH
• kationtové kanály TRPV metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze cílená MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• terciární struktura proteinů MeSH
• vysoká teplota MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH