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

Abstract

The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of gamma-aminobutyric acidA receptor (GABA(A)R) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 microg/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 GABA(A)R 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 GABA(A)R 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 microg/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 GABA(A)Ralpha1-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA(A)R 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 GABA(A)R.

Regulation of the subcellular distribution and gene expression of GABAA receptor by microtubules and microfilaments in cultured brain neurons

“…Our previous study has declared that Nlinked glycosylation is required for a normal subcellular distribution of GABA A R, because tunicamycin, an inhibitor to N-linked glycosylation, down-regulated the intracellular receptors [Lin et al, 1998]. The different responses of GABA A R to colchicine and tunicamycin indicate that microtubules and the glycosylation process of neurons probably participate in diverse regulatory mechanisms for the subcellular expression of GABA A R. After the treatment with colchicine, the distribution of NF-immunoreactivity became somewhat disorganized in the neurons.…”

“…Immunocytochemical staining was carried out as described before [Yin and Fan, 1990;Lin et al, 1998]. The cultures kept on 35 mm dishes were ®xed in 4% paraformaldehyde at RT for 20 min.…”

“…In situ hybridization cytochemistry was performed on the cultures as depicted previously [Lin et al, 1998]. The neurons were ®xed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 20 min at RT.…”

“…Based on molecular biological studies, there are at least 20 distinct subunits, a 1±6 , b 1±4 , g 1±4 , d, E, p, and r 1±3 for the composition of a presumed hetero-pentameric GABA A R [Rabow et al, 1995;Barnard et al, 1998].We have previously demonstrated that regular glycosylation of GABA A R is key to preserving a steady total number, and constant cell surface and intracellular fractions of the receptor binding sites in cultured brain neurons. It appears that the receptor protein passes through the Golgi apparatus for maturation of its oligosaccharides [Yin, 1992;Yin and Yang, 1992;Lin et al, 1998]. Moreover, cytoskeletal ß…”