Rapid turnover of the "functional" Na(+)-Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach.
Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na(+)-Ca2+ exchanger (NCX1) to correlate suppression of the Na(+)-Ca2+ exchange function with down-regulation of NCX1 protein expression. In a de-novo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in whole-cell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.
Egger
Marcel
M
University of Bern, Department of Physiology, Bühlplatz 5, CH-3012 Bern, Switzerland. egger@pyl.unibe.ch
Porzig
Hartmut
H
Niggli
Ernst
E
Schwaller
Beat
B
D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D032383:Myocytes, Cardiac / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D020319:Oligodeoxyribonucleotides, Antisense; D019831:Sodium-Calcium Exchanger