Identification of the GABA_A Receptor α3 Subunit in the IMR‐32 Neuroblastoma Cell Line

Abstract

A previous report has described the presence of f‐[35S]‐ butylbicyclophosphorothionate binding sites and GABA‐gated CI flux in the human neuroblastoma IMR‐32 cell line. We now report the further characterisation of this binding site and, even more important, the identification of the GABAA receptor α3 subunit expressed in these cells. Cell membranes prepared from IMR‐32 cells were screened by immunoblotting for reactivity with various GABAA receptor a subunit‐specific antibodies. Of these, only anti‐Cys α3 454‐467 antibodies recognised specifically and in a dose‐dependent manner an immunoreactive band. This Mr58,000 immunoreactive species and the N‐deglycosylated derivatives were both coincident with the respective homologues found in both calf cerebral cortex membranes and purified receptor preparations. This is the first report of the identification of a specific GABAA receptor subunit expressed in a human cell line, and it therefore provides a convenient model for the study of receptor structure and regulation.

Developmental exposure to the organochlorine insecticide endosulfan alters expression of proteins associated with neurotransmission in the frontal cortex

“…We first assessed the general cytotoxicity of endosulfan on the IMR‐32 neuroblastoma cell line. This cell line was chosen as it has been shown to express GABAergic receptors (Anderson et al, ; Noble et al, ; Sapp and Yeh, ). Given that endosulfan has been demonstrated to be a GABA A receptor antagonist, these cells serve as an effective in vitro model to evaluate endosulfan toxicity on neuronal cells.…”

“…In order to gain an understanding of the general neurotoxicity of endosulfan on neuronal cells we first evaluated its impact on cell viability using the IMR‐32 human neuroblastoma cell line that expresses measurable and functional levels of GABA A receptor (Anderson et al, ; Noble et al, ; Sapp and Yeh, ). Having this model was important as endosulfan specifically targets and inhibits this receptor, thus providing us with a unique in vitro model that more appropriately mirrors the biological processes of mammalian neurons being affected by endosulfan.…”

“…Given that endosulfan has been shown to target and inhibit GABA A receptors, the IMR‐32 neuroblastoma cell line was chosen to investigate the neurotoxic potential of endosulfan due to their documented expression of GABA A receptors and response to other GABAergic compounds, in vitro (Anderson et al, ; Noble et al, ; Sapp and Yeh, ). Cells were cultured in DMEM F12 media supplemented with 100 units/mL penicillin, 100 units/mL streptomycin, and 10% fetal bovine serum.…”

GABA, Selank, and Olanzapine Affect the Expression of Genes Involved in GABAergic Neurotransmission in IMR-32 Cells

“…We assessed changes in the expression of 84 genes involved in the functioning of the GABAergic system and in the processes of neurotransmission in the culture of neuroblastoma IMR-32 cells. It was shown that this cell line expresses predominantly functional GABA A receptors (Anderson et al, 1993 ; Noble et al, 1993 ; Sapp and Yeh, 2000 ), and therefore was chosen for our study. As test substances, in addition to Selank, we selected the primary ligand of the GABA A receptor, GABA, and the atypical antipsychotic, olanzapine, which has an affinity for the serotonin 5-HT 2 -receptor.…”

“…To test the hypothesis of a possible effect of Selank through the regulation of the activity of GABA A receptors, we studied the changes in expression of 84 genes involved in neurotransmission in the IMR-32 cell line in response to Selank. The human neuroblastoma cell line, IMR-32, was chosen for study because these cells express functional GABA A receptors (Anderson et al, 1993 ; Noble et al, 1993 ; Sapp and Yeh, 2000 ). To detect the effects associated with the action on the GABA A receptor, we also conducted analysis of the changes in gene expression in response to GABA, a major GABA A receptor ligand, and olanzapine, which is an atypical benzodiazepine that has the most pronounced affinity for 5-HT 2 receptors (Bymaster et al, 1996 ).…”