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J. Biol. Chem., Vol. 283, Issue 47, 32691-32703, November 21, 2008

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The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions^*

Yuanyuan Y. Wang, Rui B. Chang, Hang N. Waters, David D. McKemy, and Emily R. Liman¹

From the Department of Biological Sciences, Section of Neurobiology and Program in Neuroscience, University of Southern California, Los Angeles, California 90089

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.

Received for publication, May 9, 2008 , and in revised form, September 4, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health, NIDCD, Grant 004564 (to E. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–7.

¹ To whom correspondence should be addressed: Section of Neurobiology, University of Southern California, 3641 Watt Way, Los Angeles, CA 90089-2520. Tel.: 213-821-1454; Fax: 213-821-5290; E-mail: Liman{at}usc.edu.

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