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.
Wang
Yuanyuan Y
YY
Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2520, USA.
Chang
Rui B
RB
Waters
Hang N
HN
McKemy
David D
DD
Liman
Emily R
ER
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
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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