GABA A receptorsmediatetheactionofmanyclinicallyimportantdrugsinteractingwithdifferentbindingsites.Forsomepotentialbindingsites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular ␣1ϩ␤3Ϫ interface, which is homologous to the benzodiazepine binding site at the ␣1ϩ␥2Ϫ interface. On screening of Ͼ100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3Ϫc]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at ␣1␤3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at ␣1␤3␥2 and ␣1␤3 receptors. By mutating pocket-forming amino acid residues at the ␣1ϩ and the ␤3Ϫ side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethiosulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect of CGS 9895. The inhibition was not caused by a general inactivation of GABA A receptors, because the GABA-enhancing effect of ROD 188 or the steroid ␣-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the ␣ and ␤ subunit types forming the interface. CGS 9895thusrepresentsthefirstprototypeofdrugsmediatingbenzodiazepine-likemodulatoryeffectsviathe␣ϩ␤ϪinterfaceofGABA A receptors. Sincesuchbindingsitesarepresentat␣␤,␣␤␥,and␣␤␦receptors,suchdrugswillhaveamuchbroaderactionthanbenzodiazepinesandmight become clinical important for the treatment of epilepsy.

BACKGROUND AND PURPOSEGABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo [4,3-c]quinolin-3(5H)-one) elicits a strong and subtype-dependent enhancement of GABA-induced currents via a novel drug-binding site at extracellular ax+by-(x = 1-6, y = 1-3) interfaces. Here, we investigated 16 structural analogues of CGS 9895 for their ability to modulate GABA-induced currents of various GABAA receptor subtypes. EXPERIMENTAL APPROACHRecombinant GABAA receptor subtypes were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. KEY RESULTSMost of the compounds investigated were able to modulate GABA-induced currents of ab and abg receptors to a comparable extent, suggesting that the effect of these drugs is not dependent on the benzodiazepine site of GABAA receptors. Steric hindrance experiments demonstrated that these compounds exert their action predominantly via the ax+by-(x = 1-6, y = 1-3) interfaces. Whereas some compounds are unselectively modulating a broad range of receptor subtypes, other compounds feature remarkable functional selectivity for the a6b3g2 receptor, or behave as null modulators at some receptor subtypes investigated. CONCLUSION AND IMPLICATIONSPyrazoloquinolinones and pyrazolopyridinones represent the first prototypes of drugs exerting benzodiazepine-like modulatory effects via the a+b-interface of GABAA receptors. The discovery of modulators with functional subtype selectivity at this class of binding sites provides a highly useful tool for the investigation of a6b2/3g2 receptor function, and may lead to novel therapeutic principles. LINKED ARTICLE

Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABAA receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np(-/-)) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np(-/-) hippocampal cultures or in mature CA1 and DG wild-type (Np(+/+)) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np(-/-) neurons or in mature Np65-Fc-treated Np(+/+) neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np(-/-) cultures. Furthermore, GABAA receptor composition was altered at inhibitory synapses in Np(-/-) neurons as the α1 to α2 GABAA receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np(-/-) neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.

GABAA receptors are ligand‐gated chloride channels composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to a multiplicity of GABAA receptor subtypes with distinct subunit composition; regional, cellular and subcellular distribution; and pharmacology. Most of these receptors are composed of two α, two β and one γ2 subunits. GABAA receptors are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anaesthetics and convulsants. Whereas GABA acts at the two extracellular β+α‐ interfaces of GABAA receptors, the allosteric modulatory benzodiazepines interact with the extracellular α+γ2‐ interface. In contrast, barbiturates, neuroactive steroids and anaesthetics seem to interact with solvent accessible pockets in the transmembrane domain. Several benzodiazepine site ligands have been identified that selectively interact with GABAA receptor subtypes containing α2βγ2, α3βγ2 or α5βγ2 subunits. This indicates that the different α subunit types present in these receptors convey sufficient structural differences to the benzodiazepine binding site to allow specific interaction with certain benzodiazepine site ligands. Recently, a novel drug binding site was identified at the α+β‐ interface. This binding site is homologous to the benzodiazepine binding site at the α+γ2‐ interface and is thus also strongly influenced by the type of α subunit present in the receptor. Drugs interacting with this binding site cannot directly activate but only allosterically modulate GABAA receptors. The possible importance of such drugs addressing a spectrum of receptor subtypes completely different from that of benzodiazepines is discussed.

␥-Aminobutyric acid type A receptors (GABA A Rs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X 2 receptors (P2X 2 Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABA A Rs. Here, we investigated a possible "dynamic" interaction between GABA A Rs and P2X 2 Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X 2 Rs forms a transient complex with GABA A Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X 2 Rs and GABA A Rs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X 2 Rs results in a Ca 2؉ -dependent as well as an apparently Ca 2؉ -independent increase in the mobility and an enhanced degradation of GABA A Rs, whereas P2X 2 Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X 2 Rs and GABA A Rs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA A Rs.GABA A Rs 2 are the major inhibitory transmitter receptors in the central nervous system and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. They are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. The majority of these receptors are composed of one ␥, two ␣, and two ␤ subunits (1-3). GABA A Rs are widely distributed in the brain (4, 5) and spinal cord (6). Clusters of these receptors can be found at inhibitory synapses mediating phasic inhibition but also at extrasynaptic locations where they are mediating tonic inhibition (7). Using single particle tracking (SPT), it has been demonstrated that most neurotransmitter receptors, including GABA A Rs, are exchanged between synaptic and extrasynaptic domains by lateral diffusion (8 -10). The lateral mobility of receptors can be modulated by interaction with scaffolding molecules such as gephyrin for GABA A (7, 11) and glycine receptors (12, 13). In addition, receptor insertion and removal are considered major determinants in the regulation of receptor number at the cell surface and the strength of GABAergic transmission (14, 15).The P2X receptor superfamily includes seven different subunits (P2X 1 -P2X 7 ) (16). P2X 2 subunits mainly form homotrimeric receptors but also assemble with P2X 3 subunits to form heterotrimeric P2X 2/3 receptors (P...