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

Abstract

The complete cDNA sequence of the tilapia cardiac Na ؉ /Ca 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 (ϳ81%) and mammalian NCX1.1 (ϳ73%), 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 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 Ca 2؉ 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 ϳ7 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.

Gene Structure Evolution of the Na+-Ca2+ Exchanger (NCX) Family

“…It has been recently demonstrated that NCX4 is expressed in the zebrafish [21] but characterization of its Na + /Ca 2+ exchange and tissue localization requires exploration. As for the duplicate NCX genes in teleosts, only one NCX1a from the zebrafish [19] and three NCX1b have been cloned and demonstrated to be functional in cardiac tissue in zebrafish [19], trout [17] and tilapia [18]. Zebrafish is the only teleost in which both NCX1 have been cloned from cardiac ( NCX1h/NCX1b ) [GenBank:AY934775] and neural tissues ( NCX1n/NCX1a ) [GenBank:AY934776] [19].…”

“…Expression levels generally correlate with the perceived importance of transmembrane Ca 2+ flux in a particular cell type (i.e., high in cardiac, neuronal, and kidney tissue but relatively low in liver tissue). An NCX1 homolog has been cloned and characterized in several teleosts including trout [17], tilapia [18], and zebrafish [19]. We recently discovered a NCX gene ( NCX4 ) that has been found only in fish species [20].…”

Sarcolemmal ion currents and sarcoplasmic reticulum Ca2+content in ventricular myocytes from the cold stenothermic fish, the burbot(Lota lota)

“…This is especially true when one considers that the charge transferred by the L-type Ca 2+ channels given above was measured with 5·mmol·l -1 EGTA in the pipette solution, which augments Ca 2+ influx. Studies using non-teleost expression systems demonstrate that the trout NCX temperature-sensitivity is quite low (Q 10~1 .1) (Elias et al, 2001), whereas the NCX of a tropical fish species, the tilapia Oreochromis mossambicus, shows a similar temperature-sensitivity to mammals (Marshall et al, 2005). Surprisingly, the burbot I NCX shows a relatively strong temperature dependence (Q 10 =2.47) between 4°C and 11°C when recorded in native myocytes.…”

Characterization of zebrafish (Danio rerio) NCX4: a novel NCX with distinct electrophysiological properties

“…Due to the limited availability of NCX functional studies with the giant patch clamp, our sequence and functional comparisons are valid for only a few NCX homologs. These include NCX2 from the human (13), NCX1 from the dog (3), NCX3 from the dog/human (unpublished data), NCX1b from rainbow trout (3), and NCX1b from tilapia (19). Within the ␣-1 domain, NCX4a had the highest identity (at the amino acid level) at ϳ90% with dog NCX1, human NCX2, and human/dog NCX3.…”