The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an integrative molecular sensor for detecting environmental irritant compounds, endogenous proalgesic and inflammatory agents, pressure, and temperature. Different post-translational modifications participate in the discrimination of the essential functions of TRPA1 in its physiological environment, but the underlying structural bases are poorly understood. Here, we explored the role of the cytosolic N-terminal residue Ser602 located near a functionally important allosteric coupling domain as a potential target of phosphorylation. The phosphomimetic mutation S602D completely abrogated channel activation, whereas the phosphonull mutations S602G and S602N produced a fully functional channel. Using mutagenesis, electrophysiology, and molecular simulations, we investigated the possible structural impact of a modification (mutation or phosphorylation) of Ser602 and found that this residue represents an important regulatory site through which the intracellular signaling cascades may act to reversibly restrict or "dampen" the conformational space of the TRPA1 channel and promote its transitions to the closed state.

• MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• kationtový kanál TRPA1 chemie   genetika   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace * MeSH
• proteinové domény MeSH
• serin metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• biofyzikální jevy MeSH
• fosfatidylinositol-4,5-difosfát chemie   metabolismus   MeSH
• fosfolipidy chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• kationtový kanál TRPA1 chemie   genetika   MeSH
• kinetika MeSH
• lidé MeSH
• membránové potenciály genetika   MeSH
• metabolismus lipidů genetika   MeSH
• peptidy chemie   MeSH
• sekundární struktura proteinů MeSH
• signální transdukce genetika   MeSH
• vápník chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• kationtový kanál TRPA1 * fyziologie   chemie   účinky léků   MeSH
• lidé MeSH
• metoda terčíkového zámku metody   MeSH
• paracetamol * farmakologie   chemie   metabolismus   škodlivé účinky   MeSH
• terpeny agonisté   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Transient receptor potential ankyrin 1 (TRPA1) is a temperature-sensitive ion channel activated by various pungent and irritant compounds that can produce pain in humans. Its activation involves an allosteric mechanism whereby electrophilic agonists evoke interactions within cytosolic domains and open the channel pore through an integrated nexus formed by intracellular membrane proximal regions that are densely packed beneath the lower segment of the S1-S4 sensor domain. Studies indicate that this part of the channel may contain residues that form a water-accessible cavity that undergoes changes in solvation during channel gating. We identified conserved polar residues facing the putative lower crevice of the sensor domain that were crucial determinants of the electrophilic, voltage, and calcium sensitivity of the TRPA1 channel. This part of the sensor may also comprise a domain capable of binding to membrane phosphoinositides through which gating of the channel is regulated in a state-dependent manner.

• MeSH
• alosterická regulace MeSH
• gating iontového kanálu * MeSH
• HEK293 buňky MeSH
• kationtový kanál TRPA1 chemie   fyziologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• membránové potenciály * MeSH
• molekulární modely MeSH
• mutace MeSH
• mutageneze cílená MeSH
• proteinové domény MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH