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

Abstract

Mechanisms underlying the intracellular transport of γ-aminobutyric acidA receptor (GABAAR) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and 3H-flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3-h treatment of the cells with 1 μM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABAAR density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3-h incubation with 2 μM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the 3H-FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABAAR α subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular 3H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of α1+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABAAR α1 or α2 mRNA was present in 92±2% or 94±2% of the cytochalasin D-treated neurons, both of which were higher than 71±2-74±3% of the control and colchicine-treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABAAR in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABAAR subunits. © 2001 Wiley-Liss, Inc.