Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.     

Structural link between γ-aminobutyric acid type A (GABAA) receptor agonist binding site and inner β-sheet governs channel activation and allosteric drug modulation

Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner β-sheet trigger channel activation. Homology modeling reveals that the β4-β5 linker physically connects the neurotransmitter binding site to the inner β-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor β-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.     

Synthesis of novel cognition enhancers with pyrazolo[5,1-c][1,2,4]benzotriazine core acting at γ-aminobutyric acid type A (GABAA) receptor

Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an increasing medical need. Advances in research exploring the biological mechanisms underlying learning and memory have opened new potential approaches for development of memory-enhancing therapies addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazolo[5,1-c][1,2,4]benzotriazine core to identify ligands on GABA_A receptors subtype (benzodiazepine site on GABA_A-receptor) endowed with the potential of enhancing cognition activity without the side effects usually associated with non-selective GABA_A modulators. In fact, there is much evidence that GABA_A-R (γ-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22 act as dual functional modulators of GABA_A-Rs (promnemonic and anxiolytic agents) while only compounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and 3 mg/kg, respectively).     

Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [3H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [3H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [3H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [3H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [3H]TDBzl-etomidate and [3H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA(A)R.     

New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful for studying the interaction with the HBp-3 area (hydrogen bond point area) in the benzodiazepine site on the γ-aminobutyric acid type A (GABAA) receptor

The pharmacophoric model using ADLR procedure, based on pyrazolo[5,1-c][1,2,4]benzotriazine system, studied in our laboratory, allowed us to identify the essential interaction points (HBp-1, HBp-2, and Lp-1) and the important areas for affinity modulation (HBp-3 and Lp-2) for binding recognition at benzodiazepine (Bzs) site of GABA(A) receptors (GABA(A)-Rs). In this work ADLR method is used to rationalize the affinity data of 23 new compounds and to improve the knowledge on HBp-3 area, hydrogen bond area. Among these new compounds emerge the pyrrolo derivatives (18, 25, 28, 34, and 37) for their good affinity value (14.9>K(i)(nM)>63.0). In the orientations proposed by ADLR, the NH moiety of the pyrrole ring, independently of the position in the pyrazolobenzotriazine core, fits in HBp-3 area and points out the acceptor feature of this hydrogen bond area, already known as donor area. Unexpectedly, the oxygen atom of the furane ring does not form efficient hydrogen bond with the same area, probably for an imperfect distance. The size of substituent at position 8 is important but not necessary for the receptor recognition, in fact the interdependence between the features of the 3- and 8-substituent's is again verified, (i.e., compound 20 vs 32).     

Identification of a putative DNA replication origin in the λ-aminobutyric acid receptor subunit β3 and α5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing

The region containing the GABA_A receptor β3 and α5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone λ84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage λ84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13 kb of contiguous sequence reveals several A +T-rich regions which display potential DNA unwinding. The plasmid subclones from phage λ84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABA_A receptor subunit gene cluster.