Molecular mode of action of CGS 9895 at alpha(1)beta(2)gamma(2) GABA(A) receptors

摘要

gamma-raminobutyric type A (GABA(A)) 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 alpha+/beta- interface in the extracellular domain of GABA(A) 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 GABA(A) receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. We have identified the alpha(1)Y209 residue at the alpha+/beta- 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 alpha(1) Y209 (rat sequence). Not all compounds were found to be sensitive to mutations at the alpha(1) 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.