Activation Stoichiometry and Pore Architecture of TRPA1 Probed with Channel Concatemers

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.