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

• Published in: The Journal of biological chemistry Vol. 280; no. 32; pp. 28903 - 28911
• Main Authors: Marshall, Christian R., Pan, Tien-Chien, Le, Hoa Dinh, Omelchenko, Alexander, Hwang, Pung Pung, Hryshko, Larry V., Tibbits, Glen F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.08.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Alternative Splicing; Amino Acid Sequence; Animals; Biological Transport; Calcium - metabolism; Cell Membrane - metabolism; Cloning, Molecular; DNA, Complementary - metabolism; Electrophysiology; Evolution, Molecular; Exons; Gene Expression Regulation; Molecular Sequence Data; Oocytes - metabolism; Peptides - chemistry; Phenotype; Phylogeny; Protein Isoforms; Protein Structure, Tertiary; RNA - metabolism; Sequence Homology, Amino Acid; Sodium-Calcium Exchanger - biosynthesis; Sodium-Calcium Exchanger - genetics; Temperature; Tilapia; Xenopus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M504807200

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 α-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.