The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence a promising target for treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) channel can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also by intracellular Ca(2+) or depolarizing voltages. Within the S4-S5 linker of human TRPA1, a gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested by bouts of severe upper body pain, triggered by physical stress, fasting, or cold. To clarify the structural basis for this channelopathy, we derive a structural model of TRPA1 by combining homology modeling, molecular dynamics simulations, point mutagenesis and electrophysiology. In the vicinity of N855, the model reveals inter-subunit salt bridges between E854 and K868. Using the heterologous expression of recombinant wild-type and mutant TRPA1 channels in HEK293T cells, we indeed found that the charge-reversal mutants E854R and K868E exhibited dramatically reduced responses to chemical and voltage stimuli, whereas the charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, mutation analysis of highly conserved charged residues within the S4-S5 region revealed a gain-of-function phenotype for R852E with an increased basal channel activity, a loss of Ca(2+)-induced potentiation and an accelerated Ca(2+)-dependent inactivation. Based on the model and on a comparison with the recently revealed atomic-level structure of the related channel TRPV1, we propose that inter-subunit salt bridges between adjacent S4-S5 regions are crucial for stabilizing the conformations associated with chemically and voltage-induced gating of the TRPA1 ion channel.

• MeSH
• asparagin genetika   MeSH
• elektrická stimulace MeSH
• gating iontového kanálu účinky léků   fyziologie   MeSH
• HEK293 buňky MeSH
• isothiokyanatany farmakologie   MeSH
• kationtové kanály TRP chemie   genetika   metabolismus   MeSH
• lidé MeSH
• membránové potenciály genetika   MeSH
• metoda terčíkového zámku MeSH
• molekulární modely * MeSH
• mutace genetika   MeSH
• mutageneze MeSH
• proteiny nervové tkáně chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• serin genetika   MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• vápník metabolismus   MeSH
• vápníkové kanály chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata 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