Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the ␥-aminobutyric acid, type A (GABA A ) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA A receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of ␣ 1 H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated ␣ 1 H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that ␥ 2 Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

Background:Compounds targeting the benzodiazepine binding site of the GABA A -R are widely prescribed for the treatment of anxiety disorders, epilepsy, and insomnia as well as for preanesthetic sedation and muscle relaxation. It has been hypothesized that these various pharmacological effects are mediated by different GABA A -R subtypes. If this hypothesis is correct, then it may be possible to develop compounds targeting particular GABA A -R subtypes as, for example, selective anxiolytics with a diminished side effect profile. The pyrazolo[1,5-a]-pyrimidine ocinaplon is anxioselective in both preclinical studies and in patients with generalized anxiety disorder, but does not exhibit the selectivity between α 1 /α 2 -containing receptors for an anxioselective that is predicted by studies using transgenic mice. Results:We hypothesized that the pharmacological properties of ocinaplon in vivo might be influenced by an active biotransformation product with greater selectivity for the α 2 subunit relative to α 1 . One hour after administration of ocinaplon, the plasma concentration of its primary biotransformation product, DOV 315,090, is 38% of the parent compound. The pharmacological properties of DOV 315,090 were assessed using radioligand binding studies and two-electrode voltage clamp electrophysiology. We report that DOV 315,090 possesses modulatory activity at GABA A -Rs, but that its selectivity profile is similar to that of ocinaplon. Conclusion:These findings imply that DOV 315,090 could contribute to the action of ocinaplon in vivo, but that the anxioselective properties of ocinaplon cannot be readily explained by a subtype selective effect/action of DOV 315,090. Further inquiry is required to identify the extent to which different subtypes are involved in the anxiolytic and other pharmacological effects of GABA A -R modulators.

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA A receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, a 1 H101Cb 2 c 2 , as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the a/c subunit interface, or if it occurs in both a 1 subunits, exclusively reaction at the a/c subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 lM GABA but only partially in its absence or in the presence of 10 lM GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state. Macdonald and Olsen 1994;Davies et al. 1997;Whiting et al. 1999;Sieghart and Sperk 2002). The large extracellular N-terminal part is implicated in receptor assembly and formation of binding sites for agonist and benzodiazepines. Three transmembrane-spanning domains TM1-TM3 are connected with small loops while TM3 is connected to TM4 by a large cytoplasmic loop implicated in receptor trafficking and regulation by second messenger systems (Kittler and Moss 2003). The TM2 domain is lining the channel pore (Xu and Akabas 1996;Goren et al. 2004). abc GABA A receptors harbour two agonist binding sites (Sigel et al. 1992;Amin and Weiss 1993;Smith and Olsen 1994;Westh-Hansen et al. 1997, 1999Boileau et al. 1999;Wagner and Czajkowski 2001) and a binding site for allosteric modulators of the benzodiazepine type (Wieland et al. 1992;Amin et al. 1997;Buhr and Sigel 1997;Buhr et al. 1997a;Buhr et al. 1997b;Teissere and Czajkowski 2001;Sigel 2002). Binding of GABA to agonist sites is coupled to opening of the channel pore, which then allows flow of Cl ) ions down their electro-chemical gradient. Abbreviations used: TM1-4, transmembrane-spanning domains 1-4; NCS-compound, 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one; Ro15-1788, benzodiazepine antagonist.

GABA mediates most inhibitory synaptic transmission in the adult vertebrate CNS by activating type‐A GABA receptors that contain an integral ion channel and type‐B GABA receptors that are G‐protein coupled. GABA A receptors have been a rich target for the development of therapeutics for treatment of anxiety disorders, convulsive disorders, sleep disturbances, and for the induction of anesthesia. GABA A receptors are composed of five membrane‐spanning subunits, selected from eight subunit subtypes (α, β, γ, δ, η, ρ, π, and θ) many of which contain multiple isoforms yielding at least 21 distinct subunit variants. These variations in subunit composition can have profound effects upon the functionality, pharmacology, and subcellular distribution of receptor subtypes. This chapter focuses on the relationship between receptor architecture and pharmacology of a large number of clinically relevant compounds such as benzodiazepines, barbiturates, anesthetics, neurosteroids and alcohols.

Positive allosteric modulation of the GABA A receptor (GABA A R) via the benzodiazepine recognition site is the mechanism whereby diverse chemical classes of therapeutic agents act to reduce anxiety, induce and maintain sleep, reduce seizures, and induce conscious sedation. The binding of such therapeutic agents to this allosteric modulatory site increases the affinity of GABA for the agonist recognition site. A major unanswered question, however, relates to how positive allosteric modulators dock in the 1,4-benzodiazepine (BZD) recognition site. In the present study, the X-ray structure of an acetylcholine binding protein from the snail Lymnea stagnalis and the results from site-directed affinity-labeling studies were used as the basis for modeling of the BZD binding pocket at the ␣ 1 /␥ 2 subunit interface. A tethered BZD was introduced into the binding pocket, and molecular simulations were carried out to yield a set of candidate orientations of the BZD ligand in the binding pocket. Candidate orientations were refined based on known structureactivity and stereospecificity characteristics of BZDs and the impact of the ␣ 1 H101R mutation. Results favor a model in which the BZD molecule is oriented such that the C5-phenyl substituent extends approximately parallel to the plane of the membrane rather than parallel to the ion channel. Application of this computational modeling strategy, which integrates sitedirected affinity labeling with structure-activity knowledge to create a molecular model of the docking of active ligands in the binding pocket, may provide a basis for the design of more selective GABA A R modulators with enhanced therapeutic potential.