Downregulation and subcellular redistribution of the γ-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons

• Published in: Journal of cellular biochemistry 1998-07, Vol.70 (1), p.38-48
• Main Authors: Lin, Tzu-Yung, Wang, Seu-Mei, Yin, Hsiang-Shu
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Brain - cytology; Brain - drug effects; Brain - metabolism; Cells, Cultured; Chick Embryo; Down-Regulation - drug effects; GABAA receptor; galactosylation; immunoblotting; immunocytochemistry; in situ trypsinization; mannosylation; Molecular Weight; N-glycosylation; Neurons - drug effects; Neurons - metabolism; Oligonucleotide Probes; Radioligand Assay; radioligand binding; Receptors, GABA-A - genetics; Receptors, GABA-A - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Subcellular Fractions; Tunicamycin - pharmacology
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/(SICI)1097-4644(19980701)70:1<38::AID-JCB5>3.0.CO;2-4

The significance of N‐linked glycosylation and oligosaccharide processing was examined for the expression of γ‐aminobutyric acidA receptor (GABAAR) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 μg/ml of tunicamycin for 24 h blocked the binding of 3H‐flunitrazepam and 3H‐muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABAAR immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by 3H‐flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N‐glycosidase F digestion, indicating the blockade of N‐linked glycosylation of GABAAR by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin‐treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of 3H‐sugars and 3H‐leucine. Nevertheless, treatment with castanospermine or swainsonine (10 μg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABAAR α1‐subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABAAR by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N‐linked glycosylation may be important for the functional expression and intracellular transport of GABAAR. J. Cell. Biochem. 70:38–48, 1998. © 1998 Wiley‐Liss, Inc.