Primary cultures of cardiac muscle cells as models for investigation of protein glycosylation.
Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells. Exposure of the cells up to 6 micrograms/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (Bmax) to 46.0 +/- 17.2% of the untreated control. Similar effects were observed after enzymatic deglycosylation using N-glycosidase F (PNGase F). The results suggest that a posttranslational modification of parts of the cardiac L-type Ca+2 channel by N-glycosylation is an important determinant for the binding of Ca+2 antagonists of the DHP-type to the alpha 1 subunit which itself is not glycosylated. The results suggest a participation of N glycosylation in the assembling of the subunits to the functional channel and/or its turnover. However, a possible effect of tunicamycin on the expression of the Ca channel as an alternative mechanism cannot be excluded.
Institute of Biochemistry and Molecular Physiology, University of Potsdam, Berlin, Germany.
U
U
Henning
W P
WP
Wolf
M
M
Holtzhauer
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D004095:Dihydropyridines / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014415:Tunicamycin / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D011506:Proteins; D014415:Tunicamycin; C038806:1,4-dihydropyridine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase