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

Abstract

Mechanisms underlying the intracellular transport of gamma-aminobutyric acid(A) receptor (GABA(A)R) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and (3)H-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 microM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABA(A)R 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 microM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the (3)H-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 GABA(A)R alpha subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular (3)H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of alpha(1)+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 GABA(A)R alpha(1) or alpha(2) 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 GABA(A)R in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABA(A)R subunits.

Acrylamide disturbs the subcellular distribution of GABAA receptor in brain neurons

“…It has been suggested that acrylamide interferes with axonal and intradendritic transport by acting on microtubules as well as MAPs [Lapadula et al, 1989;Sager, 1989;Chauhan et al, 1993]. In our previous study, the colchicine treatment likewise caused an enhanced intracellular level of GABA A R in the neurons [Ho et al, 2001]. Although colchicine did not seem to significantly decrease the tubulin content in the neuronal perikarya, the depolymerization of microtubules evidently interfered intracellular transport to some extent [Ho et al, 2001].…”

“…In our previous study, the colchicine treatment likewise caused an enhanced intracellular level of GABA A R in the neurons [Ho et al, 2001]. Although colchicine did not seem to significantly decrease the tubulin content in the neuronal perikarya, the depolymerization of microtubules evidently interfered intracellular transport to some extent [Ho et al, 2001].…”

“…In addition, we have previously demonstrated increased intracellular and decreased cell surface proportions of the GABA A R in cultured brain neurons, after the destruction of microtubules with colchicine, as revealed by using in situ trypsinization and binding assay with 3 H-flunitrazepam for the benzodiazepine modulator sites [Ho et al, 2001]. Thus, it is suggested that the microtubular system participates in the intracellular transport of the receptor because microtubules are key in neural growth and intracellular transport of proteins [Aliva, 1992;Hirokawa, 1998].…”

“…Thus, it is suggested that the microtubular system participates in the intracellular transport of the receptor because microtubules are key in neural growth and intracellular transport of proteins [Aliva, 1992;Hirokawa, 1998]. Furthermore, the organization of immunostained neurofilaments was also somewhat disturbed by colchicine [Ho et al, 2001]. Neurofilaments are believed to participate in maintaining the cell shape and interact with other cytoskeletal components such as microtubules [Robinson and Anderton, 1988].…”

GABAAReceptor-Associated Protein Regulates GABAAReceptor Cell-Surface Number inXenopus laevisOocytes

“…It is feasible that oocytes express sufficient quantities of machinery, including microtubules for formation of receptor clusters, because disruption of microtubules has been shown to alter clustering and properties of GABA A receptors expressed in oocytes (Whatley et al, 1994). Accumulated evidence indicates that the cytoskeleton is essential for clustering GABA A receptors (Chen et al, 2000;Ho et al, 2001;Petrini et al, 2003). It reminds us that a reduction in clustering state or misdistribution of GABA A receptors by nocodazole or colchicine, drugs disrupting the polymerization of microtubules, might work by interrupting the interaction between polymerized microtubules and receptor-associated proteins, such as GABARAP and gephyrin.…”