Temperature dependence of the cardiac sodium calcium exchanger

Calcium (ca2') is a ubiquitous and highly versatile intracellular messenger as exemplified by the numerous mechanisms nature has evolved to facilitate ca2' transport. One of these mechanisms is ~ a ' / ~ a ~ ' exchange. The ~ a ' / ~ a ~ ' exchanger (NCX) is an integral membrane protein that catalyzes the counter-transport of Na' for ca2' ions. NCX is expressed in a wide variety of tissues throughout a diverse group of organisms. NCX expression is especially high in the heart where NCX mainly functions to extrude ca2' from the cytosol to allow relaxation. Cardiac function in ectotherms, such as the trout, is distinguished by its ability to maintain adequate contractility in temperatures that are cardioplegic to mammals. Using cloned trout NCX-TR1.O and mammalian NCX1.l expressed in Xenopus oocytes, we measured NCX mediated currents and showed that the NCX-TRl .O isoform is relatively insensitive to temperature compared with NCX1.l. Furthermore, our results indicate that this phenomenon is intrinsic to the NCX protein itself and is not due to differences in regulatory properties between isoforms. Using chimeras, we determined that the region of the NCX protein responsible for this differential temperature dependence is located within the N-terminal transmembrane domain of the protein. However, further chimeric studies within this region have produced equivocal results. To aid in generation of hypotheses as to which amino acids are likely to confer NCX temperature dependence, we employed bioinformatics and comparative analyses. Cloning of tilapia NCX-TL1.O provided a useful comparative model to NCX-TR1 .O, since tilapia share a close phylogenetic relationship with trout while adapted to live in warm waters. Despite high overall sequence identity with NCX-TR1.0, we found NCX-TL 1.0 to have mammalian like temperature dependence. Coupled with measurements using squid and fruit fly NCX isoforms and subsequent sequence analyses, these comparative studies provided insight into the relationship between NCX genotype and temperature phenotype. Finally, data mining of genomic data yielded 13 new NCX sequences, including a fourth NCX gene member that is present only in fish and amphibian genomes. Phylogenetic and evolutionary analyses indicate three serial gene duplication events occurred early in the evolution of vertebrates, giving rise to the four members of the NCX gene family.