Structure In 2016, cryo-electron microscopy was employed to obtain a three-dimensional structure of TRPA1. This work revealed that the channel assembles as a homotetramer, and possesses several structural features that hint at its complex regulation by irritants, cytoplasmic second messengers (e.g., calcium), cellular co-factors (e.g., inorganic anions like polyphosphates), and lipids (e.g., PIP2). Most notably, the site of covalent modification and activation for electrophilic irritants was localized to a tertiary structural feature on the membrane-proximal intracellular face of the channel, which has been termed the 'allosteric nexus', and which is composed of a cysteine-rich linker domain and the eponymous TRP domain. Breakthrough research combining cryo-electron microscopy and electrophysiology later elucidated the molecular mechanism of how the channel functions as a broad-spectrum irritant detector. With respect to electrophiles, which activate the channel by covalent modification of two cysteines in the allosteric nexus, it was shown that these reactive oxidative species act step-wise to modify two critical cysteine residues in the allosteric nexus. Upon covalent attachment, the allosteric nexus adopts a conformational change that is propagated to the channel's pore, dilating it to permit cation influx and subsequent cellular depolarization. With respect to activation by the second messenger calcium, the structure of the channel in complex with calcium localized the binding site for this ion and functional studies demonstrated that this site controls the various different effects of calcium on the channel – namely potentiation, desensitization, and receptor-operation. Clinical significance In 2008, it was observed that caffeine suppresses activity of human TRPA1, but it was found that mouse TRPA1 channels expressed in sensory neurons cause an aversion to drinking caffeine-containing water, suggesting that the TRPA1 channels mediate the perception of caffeine. TRPA1 has also been implicated in airway irritation by cigarette smoke, cleaning supplies and in the skin irritation experienced by some smokers trying to quit by using nicotine replacement therapies such as inhalers, sprays, or patches. A missense mutation of TRPA1 was found to be the cause of a hereditary episodic pain syndrome. A family from Colombia suffers from debilitating upper-body pain starting in infancy that is usually triggered by fasting or fatigue (illness, cold temperature, and physical exertion being contributory factors). A gain-of-function mutation in the fourth transmembrane domain causes the channel to be overly sensitive to pharmacological activation. Metabolites of paracetamol (acetaminophen) have been demonstrated to bind to the TRPA1 receptors, which may desensitize the receptors in the way