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J. Biol. Chem., Vol. 282, Issue 16, 12176-12185, April 20, 2007

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Relocalization of STIM1 for Activation of Store-operated Ca²⁺ Entry Is Determined by the Depletion of Subplasma Membrane Endoplasmic Reticulum Ca²⁺ Store^*

Hwei Ling Ong, Xibao Liu¹, Krasimira Tsaneva-Atanasova¹, Brij B. Singh^¶, Bidhan C. Bandyopadhyay, William D. Swaim, James T. Russell^||, Ramanujan S. Hegde^**, Arthur Sherman, and Indu S. Ambudkar²

From the Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, the ^¶Department of Biochemistry and Molecular Biology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58203, the ^**Cell Biology and Metabolism Branch and ^||Microscopy and Imaging Core, NICHD, National Institutes of Health, Bethesda, Maryland 20892, and the Laboratory of Biological Modeling, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca²⁺ entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca²⁺ depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 µM thapsigargin (Tg) induced substantial depletion of intracellular Ca²⁺ stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca²⁺ depletion but similar SOCE. SOCE was confirmed by measuring I_SOC in addition to Ca²⁺, Mn²⁺, and Ba²⁺ entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 µM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 µM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca²⁺] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.

Received for publication, October 5, 2006 , and in revised form, January 16, 2007.

^* 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 These authors contributed equally to this work.

² To whom correspondence should be addressed: Bldg. 10, Rm. 1N-113, 10 Center Dr., National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5298; Fax: 301-402-1228; E-mail: indu.ambudkar{at}nih.gov.

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