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J. Biol. Chem., Vol. 283, Issue 12, 8014-8022, March 21, 2008

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Dynamic Coupling of the Putative Coiled-coil Domain of ORAI1 with STIM1 Mediates ORAI1 Channel Activation^*

Martin Muik¹², Irene Frischauf¹, Isabella Derler¹³, Marc Fahrner, Judith Bergsmann, Petra Eder, Rainer Schindl, Clemens Hesch, Bernhard Polzinger, Reinhard Fritsch, Heike Kahr, Josef Madl², Hermann Gruber, Klaus Groschner, and Christoph Romanin⁴

From the Institute of Biophysics, University of Linz, A-4040 Linz, Austria and the Department of Pharmacy, University of Graz, A-8010 Graz, Austria

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca²⁺ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca²⁺ store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca²⁺ inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.

Received for publication, October 29, 2007 , and in revised form, January 9, 2008.

^* This work was supported by Austrian Science Foundation Project P18280 [GenBank] (to K. G.) and Projects P16537 [GenBank] and P18169 [GenBank] as well as Subproject 11 within W1201 (to C. R.). 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. S1-S3.

¹ These authors contributed equally to this work.

² Graduate students within the Ph. D. Program W1201 "Molecular Bioanalytics" from the Austrian Science Foundation.

³ Graduate student with a scholarship from the Austrian Academy of Sciences.

⁴ To whom correspondence should be addressed: Inst. of Biophysics, University of Linz, A-4040 Linz, Austria. Tel.: 43-732-2468-9272; Fax: 43-732-2468-9280; E-mail: christoph.romanin{at}jku.at.

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