DOWN-REGULATION AND SUBCELLULAR REDISTRIBUTION OF THE GAMMA-AMINOBUTYRIC ACID(A) RECEPTOR-INDUCED BY TUNICAMYCIN IN CULTURED BRAIN NEURONS

The significance of N-linked glycosylation and oligosaccharide process ing was examined for the expression of gamma-aminobutyric acid(A) rece ptor (GABA(A)R) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 mu g/ml of tunicamycin for 24 h blocked the binding of H-3-flunitrazepam and H-3-muscimol, probes for the benz odiazepine and GABA sites on the receptor, by about 20% and 28%, respe ctively. The loss of ligand binding was due to a reduction in the numb er 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 GABA(A)R immunoreactivi ty 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 H-3-flunitrazepam binding. The molecular weight of the receptor subuni t protein was lowered around 0.5 kDa after tunicamycin treatment, in a ccordance with that following N-glycosidase F digestion, indicating th e blockade of N-linked glycosylation of GABA(A)R by tunicamycin. Moreo ver, intense inhibitions of 91% and 44%, respectively, were detected t o the general galactosylation and mannosylation in the tunicamycin-tre ated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of H-3-sugars and H-3-leucine. Nevertheles s, treatment with castanospermine or swainsonine (10 mu g/ml, 24 h), i nhibitors to maturation of oligosaccharides, failed to produce signifi cant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABA(A)R alpha(1)-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA(A)R by prod ucing irregularly glycosylated receptors and modifying their localizat ion. Both galactosylation and mannosylation during the process of N-li nked glycosylation may be important for the functional expression and intracellular transport of GABA(A)R. (C) 1998 Wiley-Liss, Inc.