J Neurochem 2016 Sep 01;1385:722-30. doi: 10.1111/jnc.13711.

Molecular mode of action of CGS 9895 at α1 β2 γ2 GABAA receptors.

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γ-aminobutyric type A (GABAA ) receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics, and anesthetics. Previously, pyrazoloquinoline 2-p-methoxyphenylpyrazolo [4,3-c] quinolin-3(5H)-one (CGS 9895) was described as a positive allosteric modulator acting through the α+/β- interface in the extracellular domain of GABAA receptors. The localization of the binding site was based on a steric hindrance approach, rather than on direct effects of point mutations. In this study we further characterized modulation by this compound which seems to have multiple sites of action. We investigated GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. We have identified the α1 Y209 residue at the α+/β- interface as a key residue for CGS 9895 modulation. In addition, the interaction between this residue and various structural analogs was characterized, allowing tentative positioning of CGS 9895 versus α1 Y209 (rat sequence). Not all compounds were found to be sensitive to mutations at the α1 Y209 residue. In addition, the interaction of CGS 9895 with flurazepam was characterized. Flurazepam is hypothesized to act at the same subunit interface in the extracellular domain. We also provide evidence that the GABAA receptor harbors additional modulatory sites for CGS 9895 at each of the subunit interfaces in the transmembrane domain. GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We have identified the α1 Y209 residue present at the extracellular α+/β- subunit interface as a key residue for the positive allosteric modulation of the GABAA receptor by CGS 9895. This receptor harbors additional modulatory sites for this compound at subunit interfaces in the transmembrane domain.

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Species referenced: Xenopus laevis
Genes referenced: gabarap