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J. Biol. Chem., Vol. 283, Issue 5, 2663-2674, February 1, 2008

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Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

SENSITIVITY TO REGULATORY CONFORMATIONAL CHANGE AND CELL VOLUME^*

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

To examine the structure and function of the Na-K-Cl cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl^- provided a ratiometric indicator of intracellular [Cl^-]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed cotemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

Received for publication, October 2, 2007 , and in revised form, November 27, 2007.

^* This work was supported by National Institutes of Health Grant DK47661. 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 Methods 1 and Table 1.

¹ Presented a portion of this work as a Ph.D. dissertation at the University of Konstanz. Present address: Dept. of Membrane Biophysics, Max Planck Inst. for Biophysical Chemistry, D37077 Göttingen, Germany.

² To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Yale University, P. O. 208026, New Haven, CT 06520. E-mail: biff.forbush{at}yale.edu.

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