Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties

doi:10.1016/0922-4106(90)90232-M

European Journal of Pharmacology: Molecular Pharmacology

Volume 189, Issue 1, 31 July 1990, Pages 77-88

https://doi.org/10.1016/0922-4106(90)90232-M Get rights and content

Abstract

The cloned cDNAs encoding the α1 and β1 subunits of the bovine brain GABA_A receptor have been co-transfected, using a dexamethasone-inducible promoter, into cultured hamster ovary cells, with selection to form a stable cell line. The use, alternatively, of a much stronger constitutive promoter led to cell death consequent upon high receptor density. After induction, the cells contained the α1 and β1 mRNAs. the expressed receptors showed the high-affinity binding of [³H]muscimol an3 of the GABA_A receptor channel blocker, t-butylphosphorothionate (TBPS), and the characteristic enhancement of the former by a pregnanolone. Their GABA-activated current was potentiated by the barbiturate, pentobarbitone, was reversibly blocked by bicuculline and picrotoxin, but was not enhanced by benzodiazepines. In mouse spinal cord neurons GABA activates channel openings to at least four conductance states (45, 30, 19 and 12 pS) with the 30 pS state being the most frequently observed (main) state. However, the main state of the α1/β1 GABA_A receptor was the 19 pS state. The enhancement of GABA_A receptor current by barbiturates was due to prolongation of mean channel lifetime, whereas the reduction of GABA_A receptor current by picrotoxin was due to reduction of channel opening frequency and mean channel lifetime. Stable cell lines containing subunit combinations of this receptor should provide a powerful tool for the elucidation of its channel features and control mechanisms.

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All mammalian GABA_A receptor β subunits contain a conserved consensus site for phosphorylation by a number of serine/threonine protein kinases. This site corresponds to Serine 410 of the β2 subunit and Serine 409 of the β3 subunit, each of which lies within the conserved sequence R-R-R-X-S-L-Q-K, where X = A (β1,β2 and β4) or S (β3). We have analysed the phosphorylation of the β2 and β3 subunits of the murine GABA_A receptor by expressing the large intracellular domains of these subunits as soluble fusion proteins in E. coli.
The intracellular domain of the β2 subunit was phosphorylated to high stoichiometry by both cAMP- and cGMP-dependent protein kinases, protein kinase C and Ca²⁺/calmodulin type II-dependent protein kinase in vitro. Site-directed mutagenesis identified Serine 410 as the single site within the β2 subunit phosphorylated by these four protein kinases. Using similar methodologies, Serine 409 of theβ3 subunit was shown to be a substrate for phosphorylation by these protein kinases. Serine 408 was also seen to be phosphorylated by protein kinase C and Serine 383 was phosphorylated by Ca²⁺/calmodulin type II-dependent protein kinase. Since β subunits are believed to be essential for robust GABA_A receptor expression, these results suggest a critical role for conserved phosphorylated amino acids within the β subunits in coordinating cellular regulation of GABA_A receptors via multiple protein kinases.
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We are interested in establishing stably transformed insect cell lines efficiently expressing the insect γ-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non-Drosophilid insects.
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Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties

Abstract

J. Biol. Chem.

Anal. Biochem.

Brain Res.

Neuron

J. Biol. Chem.

FEBS Lett.

Neurosci. Lett.

Cell

The structure of the GABA/benzodiazepine receptor complex with its gated ion channel

Single subunits of the GABAA receptor form ion channels with properties of the native receptor

Science

Mechanism of anion permeation through channels gated by glycine and γ-aminobutyric acid in mouse cultured spinal neurones

J. Physiol.

High-efficiency tranformation of mammalian cells by plasmid DNA

Mol. Cell. Biol.

Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease

Biochemistry

Genetic reconstruction of functional acetylcholine receptor channels in mouse fibroblasts

Science

High efficiency gene transfer into mammalian cells

Single subunits of the GABA_A receptor form ion channels with properties of the native receptor