Expression of the cardiac Na(+)-Ca2+ exchanger in insect cells using a baculovirus vector.
We constructed a recombinant baculovirus containing cardiac Na(+)-Ca2+ exchanger cDNA under control of the polyhedrin promoter. When either Sf9 or Sf21 insect cells are infected with the recombinant baculovirus, both Na(+)-Ca2+ exchanger protein and Na(+)-Ca2+ exchange activity are expressed at high level. The exchanger protein can be detected either by immunoblot or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysate. At maximal expression, the exchanger protein comprises about 3-5% of total cell protein. The Na(+)-Ca2+ exchanger can be purified by alkaline extraction of infected cells followed by elution from a Bio-Rad Prep Cell. The expressed exchanger, in contrast to the native sarcolemmal exchanger, is not glycosylated. Sf9 cells expressing the exchanger are intensely stained by anti-exchanger antibodies as observed by immunofluorescence. The expressed exchanger is predominantly in the cell plasma membrane since it is susceptible to extracellular trypsin. In 45Ca2+ flux experiments, the expressed Na(+)-Ca2+ exchange activity is about 4-fold higher than that in cultured neonatal rat heart cells. The expressed exchanger was also analyzed electrophysiologically using whole cell patch clamp techniques. The characteristics of inward exchange currents in infected Sf21 cells are very similar to those of ventricular myocytes, although of a larger magnitude.
Li
Z
Z
Department of Medicine, School of Medicine, University of California, Los Angeles 90024-1760.
Smith
C D
CD
Smolley
J R
JR
Bridge
J H
JH
Frank
J S
JS
Philipson
K D
KD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D005822:Genetic Vectors; D006031:Glycosylation; D006168:Guinea Pigs; D007313:Insecta; D007700:Kinetics; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D014357:Trypsin / Q000378:metabolism
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D004247:DNA; D012964:Sodium; D014357:Trypsin; D002118:Calcium