Abstract
The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modulated by myriad noxious stimuli that interact with multiple regions of the channel, including cysteine-reactive natural extracts from onion and garlic which modify residues in the cytoplasmic domains. The way in which TRPA1 cytoplasmic domain modification is coupled to opening of the ion-conducting pore has yet to be elucidated. The cryo-EM structure of TRPA1 revealed a tetrameric C-terminal coiled-coil surrounded by N-terminal ankyrin repeat domains (ARDs), an architecture shared with the canonical transient receptor potential (TRPC) ion channel family. Similarly, structures of the TRP melastatin (TRPM) ion channel family also showed a C-terminal coiled-coil surrounded by N-terminal cytoplasmic domains. This conserved architecture may indicate a common gating mechanism by which modification of cytoplasmic domains can transduce conformational changes to open the ion-conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal coiled-coil domains can be expressed in bacteria and maintain the ability to interact. We tested three gating regulators: temperature; the polyphosphate compound IP6; and the covalent modifier allyl isothiocyanate to determine whether they alter N- and C-terminal interactions. We found that none of the modifiers tested abolished ARD-coiled-coil interactions, though there was a significant reduction at 37˚C. We found that coiled-coils tetramerize in a concentration dependent manner, with monomers and trimers observed at lower concentrations. Our system provides a method for examining the mechanism of oligomerization of TRPA1 cytoplasmic domains as well as a system to study the transmission of conformational changes resulting from covalent modification.
Highlights
The Transient Receptor Potential Ankyrin-1 (TRPA1) ion channel is expressed in nociceptors of the peripheral nervous system [1] where it is activated by a variety of noxious chemical stimuli including electrophilic covalent modifiers[1,2,3], non-covalent compounds [4], and temperature [5,6]
The primary sequence of the human TRPA1 coiled-coiled consisting of amino acids A1036-T1078 contains no tryptophan residues and few other residues that absorb at 280 nm making it difficult to observe in standard size exclusion chromatography with absorbance detection
In this study we aimed to develop a reduced in vitro system in which we could reconstitute the interactions of the N-terminal ankyrin repeat domains (ARDs) and the C-terminal coiled-coil observed in the full length human TRPA1 structure [15]
Summary
The Transient Receptor Potential Ankyrin-1 (TRPA1) ion channel is expressed in nociceptors of the peripheral nervous system [1] where it is activated by a variety of noxious chemical stimuli including electrophilic covalent modifiers[1,2,3], non-covalent compounds [4], and temperature [5,6]. TRPA1 is involved in inflammatory signaling [7] and has become an active therapeutic target for treatment of cough [8,9], itch [9,10], and pain [10,11]. Despite the importance of TRPA1 in sensing noxious stimuli, the structural mechanisms of channel activation remain unknown. Since there are multiple channel activators, both covalent. Health: S10RR025429, P30DK017047, and P30EY001730, and the University of Washington Royalty Research Fund
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