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J. Biol. Chem., Vol. 283, Issue 22, 14938-14945, May 30, 2008

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Voltage Gating at the Selectivity Filter of the Ca²⁺ Release-activated Ca²⁺ Channel Induced by Mutation of the Orai1 Protein^*

Maria A. Spassova¹, Thamara Hewavitharana, Richard A. Fandino, Asli Kaya, Jacqueline Tanaka, and Donald L. Gill

From the Biology Department, Temple University, College of Science and Technology, Philadelphia, Pennsylvania 19122 and the Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21201

The Ca²⁺ release-activated Ca²⁺ (CRAC) channel is a plasma membrane (PM) channel that is uniquely activated when free Ca²⁺ level in the endoplasmic reticulum (ER) is substantially reduced. Several small interfering RNA screens identified two membrane proteins, Orai1 and STIM1, to be essential for the CRAC channel function. STIM1 appears to function in the PM and as the Ca²⁺ sensor in the ER. Orai1 is forming the pore of the CRAC channel. Despite the recent breakthroughs, a mechanistic understanding of the CRAC channel gating is still lacking. Here we reveal new insights on the structure-function relationship of STIM1 and Orai1. Our data suggest that the cytoplasmic coiled-coil region of STIM1 provides structural means for coupling of the ER membrane to the PM to activate the CRAC channel. We mutated two hydrophobic residues in this region to proline (L286P/L292P) to introduce a kink in the first -helix of the coiled-coil domain. This STIM1 mutant caused a dramatic inhibition of the CRAC channel gating compared with the wild type. Structure-function analysis of the Orai1 protein revealed the presence of intrinsic voltage gating of the CRAC channel. A mutation of Orai1 (V102I) close to the selectivity filter modified CRAC channel voltage sensitivity. Expression of the Orai1^V102I mutant resulted in slow voltage gating of the CRAC channel by negative potentials. The results revealed that the alteration of Val¹⁰² develops voltage gating in the CRAC channel. Our data strongly suggest the presence of a novel voltage gating mechanism at the selectivity filter of the CRAC channel.

Received for publication, March 14, 2007 , and in revised form, December 18, 2007.

^* This work was supported, in whole or in part, by National Institutes of Health Grant AI058173 to the University of Maryland School of Medicine. 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.

¹ To whom correspondence should be addressed: Dept. of Biology, Temple University, 1900 N. 12th St., Philadelphia, PA 19122. E-mail: spassova{at}temple.edu.

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