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 Ca2+ is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca2+ 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 Ca2+ permeability was greatly reduced, extracellular Ca2+ produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca2+ buffering, which allowed intracellular Ca2+ levels to become elevated upon entry through D918A channels. Application of Ca2+ 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 Ca2+ 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 Mg2+, Ba2+, and Ca2+ but potentiated only by Ba2+ and Ca2+. 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 Ca2+-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 Ca2+ influx through TRPA1 to nociceptive signaling.