Abstract
The nociceptor ion channel TRPA1 detects a wide range of hazardous chemicals, including reactive electrophiles such as cinnamaldehyde, which gate the channel allowing Na+ and Ca2+ entry. TRPA1 assembles as a tetramer, with a central pore within which an aspartate residue (D918) determines Ca2+ permeability. Here, we report that introduction of histidine at this position, D918H, makes TRPA1 channels sensitive to block by nanomolar concentration of Zn2+ and can be used to functionally tag subunits in concatemers. Concatemers with increasing numbers of D918H subunits display increasing sensitivity to Zn2+ inhibition, indicating that the four side chains at position 918 of the tetramer directly coordinate Zn2+ and other permeating divalent cations. In the published structure of TRPA1, this requires a rearrangement of the pore region which may represent the true open state of the channel. Concatemeric channels containing subunits mutated to be insensitive to reactive electrophiles (C622S) could be activated by cinnamaldehyde when as few as two subunits contained intact ligand binding sites. Activation upon liganding of just two of the four possible subunits may represent an optimal strategy to rapidly and reliably detect noxious chemicals.
Highlights
Similar to other TRP channels, a single TRPA1 channel subunit has six transmembrane helices with a pore-forming region between the 5th and 6th transmembrane domains[12,13]
TRPA1 D918H channel was strongly activated by cinnamaldehyde, and like wild-type channels was sensitive to inhibition by the TRPA1 specific blocker, A-967079, consistent with A-967079 binding site being at the lower position of the permeating pathway away from D91812 (Fig. subunit 1 (S1))
These results show that D918H makes a functional ion channel that can be activated by reactive electrophiles
Summary
Similar to other TRP channels, a single TRPA1 channel subunit has six transmembrane helices with a pore-forming region between the 5th and 6th transmembrane domains[12,13]. One channel is composed of four subunits with their pore-forming regions facing the centre that make a single ion permeation pathway. The activators of TRPA1 include a broad range of chemicals without apparent structural similarities[1], and which instead activate TRPA1 through their electrophilic properties[16,17] These electrophiles bind covalently to cysteine residues within cytoplasmic regions of the channel, resulting in a conformational change that opens the channel. By testing concatemers in which varying numbers of subunits carry the C622S mutation that renders them insensitive to activation by reactive electrophiles, we show that modification of two of the four C622 thiol groups in the channel tetramer is sufficient to open the channel
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