Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger

Isoform 1 of the cardiac Na+/Ca2+ exchanger (NCX1) is an important regulato r of cytosolic Ca2+ concentration in contraction and relaxation. Studies wi th trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7 degrees C, and this unique property is likely due to differen ces in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout (Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to h ave nine transmembrane segments. The sequences demonstrated to be the excha nger inhibitory peptide site and the regulatory Ca2+ binding site in the cy toplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1 . hTCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days curre nts were measured by the giant excised patch technique. hTCX-TR1 currents m easured at similar to 23 degrees C demonstrated Na+-dependent inactivation and Ca2+-dependent activation in a manner qualitatively similar to that for NCX1 currents.