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J. Biol. Chem., Vol. 281, Issue 8, 5017-5025, February 24, 2006

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A New Type of Non-Ca²⁺-buffering Apo(a)-based Fluorescent Indicator for Intraluminal Ca²⁺ in the Endoplasmic Reticulum^*

From the Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria

Genetically encoded Ca²⁺ indicators are outstanding tools for the assessment of intracellular/organelle Ca²⁺ dynamics. Basically, most indicators contain the Ca²⁺-binding site of a (mutated) cytosolic protein that interacts with its natural (mutated) interaction partner upon binding of Ca²⁺. Consequently, a change in the structure of the sensor occurs that, in turn, alters the fluorescent properties of the sensor. Herein, we present a new type of genetically encoded Ca²⁺ indicator for the endoplasmic reticulum (ER) (apoK1-er (W. F. Graier, K. Osibow, R. Malli, and G. M. Kostner, patent application number 05450006.1 at the European patent office)) that is based on a single kringle domain from apolipoprotein(a), which is flanked by yellow and cyan fluorescent protein at the 3'- and 5'-ends, respectively. Notably, apoK1-er does not interact with Ca²⁺ itself but serves as a substrate for calreticulin, the main constitutive Ca²⁺-binding protein in the ER. ApoK1-er assembles with calreticulin and the protein disulfide isomerase ERp57 and undergoes a conformational shift in a Ca²⁺-dependent manner that allows fluorescence resonance energy transfer between the two fluorophores. This construct primarily offers three major advantages compared with the already existing probes: (i) it resolves perfectly the physiological range of the free Ca²⁺ concentration in the ER, (ii) expression of apoK1-er does not affect the Ca²⁺ buffering capacity of the ER, and (iii) apoK1-er is not inactivated by binding of constitutive interaction partners that prevent Ca²⁺-dependent conformational changes. These unique characteristics of apoK1-er make this sensor particularly attractive for studies on ER Ca²⁺ signaling and dynamics in which alteration of Ca²⁺ fluctuations by expression of any additional Ca²⁺ buffer essentially has to be avoided.

Received for publication, August 4, 2005 , and in revised form, November 15, 2005.

^* This work was supported by the Austrian Science Funds (SFB 714, P16860 [GenBank] -B9) and the Franz-Lanyar-Stiftung at the Medical University Graz. 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, Harrachgasse 21/III, A-8010 Graz, Austria. Tel.: 43-316-380-7560; Fax: 43-316-380-9615; E-mail: wolfgang.graier{at}meduni-graz.at.

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