cDNA cloning and expression of the cardiac Na+/Ca2+ exchanger from Mozambique tilapia (Oreochromis mossambicus) reveal a teleost membrane transporter with mammalian temperature dependence.
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 ( approximately 81%) and mammalian NCX1.1 ( approximately 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(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 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 approximately 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.
Marshall
Christian R
CR
Department of Molecular Biology and Biochemistry and the Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Pan
Tien-Chien
TC
Le
Hoa Dinh
HD
Omelchenko
Alexander
A
Hwang
Pung Pung
PP
Hryshko
Larry V
LV
Tibbits
Glen F
GF
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D019143:Evolution, Molecular; D005091:Exons; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010802:Phylogeny; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis* / Q000235:genetics*; D013696:Temperature; D017210:Tilapia; D014981:Xenopus
D018076:DNA, Complementary; D010455:Peptides; D020033:Protein Isoforms; D019831:Sodium-Calcium Exchanger; D012313:RNA; D002118:Calcium