title: The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2520, USA.
authors.forename: Yuanyuan Y
authors.initials: YY
authors.lastname: Wang
authors.forename: Rui B
authors.initials: RB
authors.lastname: Chang
authors.forename: Hang N
authors.initials: HN
authors.lastname: Waters
authors.forename: David D
authors.initials: DD
authors.lastname: McKemy
authors.forename: Emily R
authors.initials: ER
authors.lastname: Liman
mesh_terms: D020816:Amino Acid Motifs; D002021:Buffers; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000502:physiology*; D004492:Edetic Acid / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D009619:Nociceptors / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D015203:Reproducibility of Results; D015398:Signal Transduction; D000074025:TRPA1 Cation Channel; D013997:Time Factors; D050051:Transient Receptor Potential Channels / Q000737:chemistry / Q000502:physiology*; D014466:Ultraviolet Rays
chemical_list: D002021:Buffers; D015220:Calcium Channels; D007477:Ions; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; D004492:Edetic Acid; D002118:Calcium
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