Stable GABAA receptor intermediates in SF-9 cells expressing ?1, ?2 and ?2 subunits

Elster, Lisbeth; Schousboe, Arne; Olsen, Richard W.

doi:10.1002/1097-4547(20000715)61:2<193::aid-jnr10>3.0.co;2-s

Cited by 5 publications

(2 citation statements)

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“…Cells were treated with drugs and harvested at 60 h post‐infection as previously described (Srinivasan et al . 1999; Elster et al . 2000).…”

Section: Methodsmentioning

confidence: 99%

“…Sf9 cells, which were grown at 278C without CO 2 supplementation, were infected with the baculovirus at a multiplicity of infection (MOI) of about 5 for each virus. Cells were treated with drugs and harvested at 60 h post-infection as previously described (Srinivasan et al 1999;Elster et al 2000). For all of the experiments involving phosphorylation drugs, 1 mm diazepam (in 0.1% ethanol) or vehicle was added to the Sf9 cells 1 h post-infection.…”

Section: Methodsmentioning

confidence: 99%

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Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms

Ali

Olsen

2001

Journal of Neurochemistry

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Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA A receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [ 3 H]¯unitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist,¯umazenil.Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immuno¯uorescence after chronic exposure to either BZ or the PKA inhibitor H-89.

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“…Cells were treated with drugs and harvested at 60 h post‐infection as previously described (Srinivasan et al . 1999; Elster et al . 2000).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms

Ali

Olsen

2001

Journal of Neurochemistry

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Assembly and intracellular trafficking of GABAA receptors

Barnes

2001

International Review of Neurobiology

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Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Elster¹,

Kristiansen²,

Pickering³

et al. 2001

Neurochemistry International

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Stable GABAA receptor intermediates in SF-9 cells expressing ?1, ?2 and ?2 subunits

Cited by 5 publications

References 58 publications

Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms

Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms

Assembly and intracellular trafficking of GABAA receptors

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

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Stable GABAA receptor intermediates in SF-9 cells expressing ?1, ?2 and ?2 subunits

Cited by 5 publications

References 58 publications

Chronic benzodiazepine treatment of cells expressing recombinant GABAA receptors uncouples allosteric binding: studies on possible mechanisms

Chronic benzodiazepine treatment of cells expressing recombinant GABAA receptors uncouples allosteric binding: studies on possible mechanisms

Assembly and intracellular trafficking of GABAA receptors

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

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Product

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Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms

Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms