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Abstract
GABA(A) receptors can be modulated by benzodiazepines, although these compounds do not directly activate or inhibit the receptors. The prototypic benzodiazepine, diazepam, potentiates responses to GABA in GABA(A) receptors that contain a gamma subunit. Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of the F-loop residues Asp(192)-Arg(197) in the GABA(A) receptor gamma(2) subunit in diazepam potentiation of the GABA response. Substitution of all of these residues with Ala and/or a residue with similar chemical properties to the wild type residue decreased the level of diazepam potentiation, and one mutation (D192A) resulted in its complete ablation. None of the mutations changed the GABA EC(50) or the [(3)H]flumazenil binding affinity, suggesting they do not affect GABA or benzodiazepine binding characteristics; we therefore propose that they are involved in the diazepam-mediated conformational change that results in an increased response to GABA. Homology models of the receptor binding pocket in agonist-bound and unbound states suggest that the F-loop is flexible and has different orientations in the two states. Considering our data in relation to these models, we find that the F-loop residues could contribute to hydrogen bond networks and hydrophobic interactions with neighboring residues that change during receptor activation.
Highlights
Benzodiazepine agonists potentiate GABAA receptor-mediated GABA currents, whereas inverse agonists inhibit GABA responses, and antagonists competitively block the action of other benzodiazepines
Rusch and Forman [2] used constitutively partially open channels to show that the classical benzodiazepine agonists diazepam and midazolam, and an inverse agonist (FG7142), altered currents through the receptors in the absence of GABA, supporting previous evidence that benzodiazepines contribute directly to channel gating by altering the open-close equilibrium of the receptor [3, 4]
Some of the machinery involved in coupling ligand binding to channel gating in the Cys-loop receptors is located at the extracellular/transmembrane interface [7,8,9,10,11], and coupling of benzodiazepine agonist binding to allosteric modulation appears to involve residues at this interface [12]
Summary
Materials—All cell culture reagents were obtained from Invitrogen, except fetal calf serum, which was from Labtech International (Ringmer, UK). [3H]Flumazenil (87.0 Ci/mmol) was from PerkinElmer Life Sciences. Cell Culture—Human embryonic kidney (HEK) 293 cells were maintained on 90-mm tissue culture plates at 37 °C and 7% CO2 in a humidified atmosphere They were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mix F-12 (1:1). Transfected HEK 293 cells were washed twice with phosphatebuffered saline and scraped into 1 ml of ice-cold HEPES buffer (10 mM, pH 7.4) with proteinase inhibitors and frozen at Ϫ20 °C. After thawing, they were washed twice with HEPES buffer and resuspended, and 50 g of cell membranes was incubated in 0.5 ml of HEPES buffer containing the benzodiazepine antagonist [3H]flumazenil.
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