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

The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific.

• MeSH
• anizotropie MeSH
• buněčná membrána chemie   metabolismus   MeSH
• kationtové kanály TRP chemie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• membránové lipidy chemie   metabolismus   MeSH
• membrány umělé * MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• proteiny nervové tkáně chemie   metabolismus   MeSH
• pufry MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• terciární struktura proteinů MeSH
• vápníkové kanály chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• změna skupenství MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH