cDNA Cloning and Expression of the Cardiac Na super(+)/Ca super(2+) Exchanger from Mozambique Tilapia (Oreochromis mossambicus) Reveal a Teleost Membrane Transporter with Mammalian Temperature Dependence

• Published in: The Journal of biological chemistry 2005-08, Vol.280 (32), p.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: Article
• Language: English
• ISSN: 0021-9258; 1083-351X

The complete cDNA sequence of the tilapia cardiac Na super(+)/Ca super(2+) 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 ( similar to 81%) and mammalian NCX1.1 ( similar to 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 alpha -repeats, the exchanger inhibitory peptide (XIP) site, and Ca super(2+)-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 Ca super(2+) and Na super(+)-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 similar to 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 plus or minus 9 and 67 plus or minus 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.