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J. Biol. Chem., Vol. 282, Issue 7, 4453-4462, February 16, 2007

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Analysis of Ion Interactions with the K⁺ -dependent Na⁺/Ca⁺ Exchangers NCKX2, NCKX3, and NCKX4

IDENTIFICATION OF THR-551 AS A KEY RESIDUE IN DEFINING THE APPARENT K⁺ AFFINITY OF NCKX2^*

Frank Visser¹, Valeria Valsecchi, Lucio Annunziato, and Jonathan Lytton²

From the Libin Cardiovascular Institute of Alberta and the Hotchkiss Brain Institute, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada and the Division of Pharmacology, Department of Neuroscience, School of Medicine, Federico II University of Naples, 80131 Naples, Italy

K⁺-dependent Na⁺/Ca²⁺ exchangers (NCKX) catalyze cytosolic Ca²⁺ extrusion and are particularly important for neuronal Ca²⁺ signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca²⁺ and Na⁺ were similar, NCKX3 and -4 displayed 40-fold higher affinities for K⁺ ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K⁺ affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca²⁺ affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K⁺ and Ca²⁺ affinities were reduced. These results indicate that the apparently low K⁺ affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K⁺ liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K⁺ binding pocket, because it was not possible to recreate the K⁺-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca²⁺ signaling.

Received for publication, November 14, 2006 , and in revised form, November 30, 2006.

^* This work was supported in part by the Canadian Institutes of Health Research (Grant FRN15035). 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 Holds postdoctoral fellowships from the Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research.

² A scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-2892; Fax: 403-283-4841; E-mail: jlytton{at}ucalgary.ca.

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