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J. Biol. Chem., Vol. 280, Issue 32, 28903-28911, August 12, 2005

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cDNA Cloning and Expression of the Cardiac Na⁺/Ca²⁺ Exchanger from Mozambique Tilapia (Oreochromis mossambicus) Reveal a Teleost Membrane Transporter with Mammalian Temperature Dependence^*

Christian R. Marshall¶, Tien-Chien Pan||, Hoa Dinh Le**, Alexander Omelchenko**, Pung Pung Hwang||, Larry V. Hryshko**, and Glen F. Tibbits

From the Department of Molecular Biology and Biochemistry and the Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada, Cardiovascular Sciences, British Columbia Research Institute for Children's & Women's Health, Vancouver, British Columbia V5Z 4H4, Canada, ^||Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, and ^**Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

The complete cDNA sequence of the tilapia cardiac Na⁺/Ca²⁺ exchanger (NCX-TL1.0) was determined. The 3.1-kb transcript encodes a protein 957 amino acids in length, with a predicted signal peptide cleaved at residue 31 and two potential N-glycosylation sites in the extracellular N terminus. Hydropathy analysis and sequence comparison predicted a mature protein with nine transmembrane-spanning segments, consistent with the structural topologies of other known mammalian and teleost NCX isoforms. Overall sequence comparison shows high identity to both trout NCX-TR1.0 (81%) and mammalian NCX1.1 (73%), and phylogenetic analyses confirmed its identity as a member of the NCX1 gene family, expressing exons A, C, D, and F in the alternative splice site. Sequence identity is even higher in the -repeats, the exchanger inhibitory peptide (XIP) site, and Ca²⁺-binding domains, which is reflected in the functional and regulatory properties of tilapia NCX-TL1.0. When NCX-TL1.0 was expressed in Xenopus oocytes and the currents were measured in giant excised patches, they displayed both positive regulation by Ca²⁺ and Na⁺-dependent inactivation in a manner similar to trout NCX-TR1.0. However, tilapia NCX-TL1.0 exhibited a relatively high sensitivity to temperature compared with trout NCX-TR1.0. Whereas trout NCX-TR1.0 currents displayed activation energies of 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 ± 9 and 67 ± 21 kJ/mol, respectively. Using comparative sequence analysis, we highlighted 10 residue positions in the N-terminal domain of the NCX that, in combination, may confer exchanger temperature dependence through subtle changes in protein flexibility. Tilapia NCX-TL1.0 represents the first non-mammalian NCX to exhibit a mammalian temperature dependence phenotype and will prove to be a useful model in defining the interplay between molecular flexibility and stability in NCX function.

Received for publication, May 2, 2005

^* This work was supported in part by Natural Sciences and Engineering Research Council of Canada Grant RGPIN 2321 (to G. F. T.) and Canadian Institutes of Health Research Grant 43873 (to L. V. H.). 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY283779.

^¶ Supported by a Ph.D. research trainee scholarship from the Michael Smith Foundation for Health Research.

Recipient of a Tier II Canada Research Chair.

Recipient of a Tier I Canada Research Chair. To whom correspondence should be addressed: Cardiac Membrane Research Laboratory, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada. Tel.: 604-291-3658; Fax: 604-291-3004; E-mail: tibbits{at}sfu.ca.

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