title: Glycosylation is required for maintenance of functional sodium channels in neuroblastoma cells.
abstract: Experimental conditions were established under which tunicamycin inhibits glycosylation by 80-90% but reduces protein biosynthesis by only 10-20% in cultured neuroblastoma cells. Growth in the presence of tunicamycin (1 micrograms/ml) reduces the number of sodium channels, as measured by high affinity saxitoxin (STX) binding to 20-28% of control values over a 60-h period without affect on the KD for STX. Neurotoxin-activated 22Na+ influx mediated by the sodium channel was similarly reduced without affect on the KD for batrachotoxin. Comparison of STX binding by intact cells or homogenates showed that tunicamycin reduces cell surface STX receptors without accumulation of an intracellular pool of binding sites. Tunicamycin caused a similar reduction in cell surface STX receptors in the presence of the lysosomal inhibitor chloroquine, suggesting that its action is not entirely due to acceleration of sodium channel degradation. The action of tunicamycin is at least partially reversible. After washout, STX receptors appear at an initial rate of approximately 1900/cell/h. Protein synthesis is required for the appearance of new sodium channels. After inhibition of sodium channel biosynthesis by either cycloheximide or tunicamycin, the number of high affinity STX receptor sites is reduced with a half-time of 26 h. Thus, at steady state, neuroblastoma cells which contain 50,000 +/- 15,000 STX receptors degrade and replace 1330 +/- 400 STX receptor sites/h. Our results show that glycosylation is an essential process in the maintenance of the normal steady state of biosynthesis and degradation of sodium channels.
mesh_terms: D029845:Amphibian Proteins; D000818:Animals; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D012530:Saxitoxin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013997:Time Factors; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; C027525:saxitoxin-binding protein, Rana catesbeiana; D014415:Tunicamycin; D012530:Saxitoxin; D002738:Chloroquine; D012964:Sodium