Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger |
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Authors: | Xue Xiao-Hua; Hryshko Larry V; Nicoll Debora A; Philipson Kenneth D; Tibbits Glen F |
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Abstract: | Isoform 1 of the cardiacNa+/Ca2+exchanger (NCX1) is an important regulator of cytosolicCa2+ concentration in contractionand relaxation. Studies with trout heart sarcolemmal vesicles haveshown NCX to have a high level of activity at 7°C, and this uniqueproperty is likely due to differences in protein structure. In thisstudy, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAPII cDNA library constructed from rainbow trout(Oncorhynchus mykiss) heart RNA. TheNCX-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 plotindicates the protein contains 12 hydrophobic segments (of which thefirst is predicted to be a cleaved leader peptide) and a largecytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have ninetransmembrane segments. The sequences demonstrated to be the exchangerinhibitory peptide site and the regulatoryCa2+ binding site in thecytoplasmic loop of mammalian NCX1 are almost completely conserved inNCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4days currents were measured by the giant excised patch technique.NCX-TR1 currents measured at ~23°C demonstratedNa+-dependent inactivation andCa2+-dependent activation in amanner qualitatively similar to that for NCX1 currents. |
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