Cloning, expression, and characterization of the trout cardiac Na(+)/Ca(2+) exchanger.
Isoform 1 of the cardiac Na(+)/Ca(2+) exchanger (NCX1) is an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation. Studies with 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 differences 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 have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca(2+) binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at approximately 23 degrees C demonstrated Na(+)-dependent inactivation and Ca(2+)-dependent activation in a manner qualitatively similar to that for NCX1 currents.
Xue
X H
XH
Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia V5A 1S6.
Hryshko
L V
LV
Nicoll
D A
DA
Philipson
K D
KD
Tibbits
G F
GF
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular; D004553:Electric Conductivity; D005260:Female; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014982:Xenopus laevis
D019831:Sodium-Calcium Exchanger