TRPA1 is a component of the nociceptive response to CO2.

In humans, high concentrations of CO(2), as found in carbonated beverages, evoke a mixture of sensations that include a stinging or pungent quality. The stinging sensation is thought to originate with the activation of nociceptors, which innervate the respiratory, nasal, and oral epithelia. The molecular basis for this sensation is unknown. Here we show that CO(2) specifically activates a subpopulation of trigeminal neurons that express TRPA1, a mustard oil- and cinnamaldehyde-sensitive channel, and that these responses are dependent on a functional TRPA1 gene. TRPA1 is sufficient to mediate responses to CO(2) as TRPA1 channels expressed in HEK-293 cells, but not TRPV1 channels, were activated by bath-applied CO(2). CO(2) can diffuse into cells and produce intracellular acidification, which could gate TRPA1 channels. Consistent with this mechanism, TRPA1 channels in excised patches were activated in a dose-dependent manner by intracellular protons. We conclude that TRPA1, by sensing intracellular acidification, constitutes an important component of the nociceptive response to CO(2).

A TRPA1-dependent mechanism for the pungent sensation of weak acids.

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions.

The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca(2+) is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca(2+) on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca(2+) permeability was greatly reduced, extracellular Ca(2+) produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca(2+) buffering, which allowed intracellular Ca(2+) levels to become elevated upon entry through D918A channels. Application of Ca(2+) to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca(2+) by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg(2+), Ba(2+), and Ca(2+) but potentiated only by Ba(2+) and Ca(2+). Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca(2+)-binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca(2+) influx through TRPA1 to nociceptive signaling.