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J. Biol. Chem., Vol. 283, Issue 28, 19265-19273, July 11, 2008

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Complex Actions of 2-Aminoethyldiphenyl Borate on Store-operated Calcium Entry^*

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709

Store-operated Ca²⁺ entry (SOCE) is likely the most common mode of regulated influx of Ca²⁺ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca²⁺ current (I_CRAC). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca²⁺ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I_CRAC-like currents in cells co-expressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than +50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca²⁺ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.

Received for publication, February 25, 2008 , and in revised form, May 15, 2008.

^* This work was supported, in whole or in part, by the National Institutes of Health NIEHS Intramural Program. 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 and 2.

¹ To whom correspondence should be addressed: NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC. Tel.: 919-541-1420; Fax: 919-541-1898; E-mail: putney{at}niehs.nih.gov.

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