Stable expression of the cardiac sodium-calcium exchanger in CHO cells.
A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchanger. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis.
Pijuan
V
V
Department of Pharmacology, School of Medicine, University of Alabama, Birmingham 35294.
Zhuang
Y
Y
Smith
L
L
Kroupis
C
C
Condrescu
M
M
Aceto
J F
JF
Reeves
J P
JP
Smith
J B
JB
D000818:Animals; D001011:Aorta; D001693:Biological Transport, Active; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D006321:Heart / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D009131:Muscle, Smooth, Vascular; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D014162:Transfection
D002132:Calcium Radioisotopes; D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D010042:Ouabain; D012964:Sodium; D008715:Methionine; D011188:Potassium; D002118:Calcium