Isabella Sarto-Jackson

The GABAA Receptor α+β− Interface: A Novel Target for Subtype Selective Drugs

GABAA receptors mediate the action of many clinically important drugs interacting with different binding sites. For some potential binding sites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular α1+β3− interface, which is homologous to the benzodiazepine binding site at the α1+γ2− interface. On screening of >100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3−c]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at α1β3 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 α1β3γ2 and α1β3 receptors. By mutating pocket-forming amino acid residues at the α1+ and the β3− 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 GABAA receptors, because the GABA-enhancing effect of ROD 188 or the steroid α-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the α and β subunit types forming the interface. CGS 9895 thus represents the first prototype of drugs mediating benzodiazepine-like modulatory effects via the α+β− interface of GABAA receptors. Since such binding sites are present at αβ, αβγ, and αβδ receptors, such drugs will have a much broader action than benzodiazepines and might become clinical important for the treatment of epilepsy.

A novel GABAA receptor pharmacology: drugs interacting with the α+β− interface

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.

Regulation of GABAA Receptor Dynamics by Interaction with Purinergic P2X2 Receptors

γ-Aminobutyric acid type A receptors (GABAARs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X2 receptors (P2X2Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABAARs. Here, we investigated a possible “dynamic” interaction between GABAARs and P2X2Rs 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 P2X2Rs forms a transient complex with GABAARs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X2Rs and GABAARs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X2Rs results in a Ca2+-dependent as well as an apparently Ca2+-independent increase in the mobility and an enhanced degradation of GABAARs, whereas P2X2Rs 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 P2X2Rs and GABAARs 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 GABAARs.

Subtype selectivity of α+β− site ligands of GABAA receptors: identification of the first highly specific positive modulators at α6β2/3γ2 receptors

GABAA 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 αx+βy− (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.

Assembly of GABAA receptors (Review)

GABAA receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABAA receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABAA receptor subtypes in the brain. The majority of GABAA receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABAA receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABAA receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABAA receptors are discussed in the light of recent publications.

Identification of amino acid residues important for assembly of GABAA receptor α1 and γ2 subunits

Comparative models of GABAA receptors composed of α1β3γ2 subunits were generated using the acetylcholine‐binding protein (AChBP) as a template and were used for predicting putative engineered cross‐link sites between the α1 and the γ2 subunit. The respective amino acid residues were substituted by cysteines and disulfide bond formation between subunits was investigated on co‐transfection into human embryonic kidney (HEK) cells. Although disulfide bond formation between subunits could not be observed, results indicated that mutations studied influenced assembly of GABAA receptors. Whereas residue α1A108 was important for the formation of assembly intermediates with β3 and γ2 subunits consistent with its proposed location at the α1(+) side of GABAA receptors, residues γ2T125 and γ2P127 were important for assembly with β3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues α1F99C, α1S106C or γ2T126C only impaired the formation of receptors at the cell surface when co‐expressed with subunits in which their predicted interaction partner was also mutated. These data are consistent with the prediction that the mutated residue pairs are located close to each other.

Spontaneous Cross-link of Mutated α1 Subunits during GABAA Receptor Assembly

γ-Aminobutyric acid, type A (GABAA) receptor α1 subunits containing a cysteine mutation at a position in the channel mouth (H109C) surprisingly formed a spontaneous cross-link with each other in receptors composed of α1H109C, β3, and γ2 subunits. Cross-linking of two α1H109C subunits did not significantly change the affinity of [3H]muscimol or [3H]Ro15-1788 binding in α1H109Cβ3γ2 receptors, but GABA displayed a reduced potency for activating chloride currents. On reduction of the disulfide bond, however, GABA activation as well as diazepam modulation was similar in mutated and wild-type receptors, suggesting that these receptors exhibited the same subunit stoichiometry and arrangement. Disulfide bonds could not be reoxidized by copper phenanthroline after having been reduced in completely assembled receptors, suggesting that cross-linking can only occur at an early stage of assembly. The cross-link of α1H109C subunits and the subsequent transport of the resulting homodimers to the cell surface caused a reduction of the intracellular pool of α1H109C subunits and a reduced formation of completely assembled receptors. The formation of α1H109C homodimers as well as of correctly assembled GABAA receptors containing cross-linked α1H109C subunits could indicate that homodimerization of α1 subunits via contacts located in the channel mouth might be one starting point of GABAA receptor assembly. Alternatively the assembly mechanism might have started with the formation of heterodimers followed by a cross-link of mutated α1 subunits at the heterotrimeric stage. The formation of cross-linked α1H109C homodimers would then have occurred independently in a separate pathway.

How to bake a brain: yeast as a model neuron.

More than 30xa0years ago Dan Koshland published an inspirational essay presenting the bacterium as a model neuron (Koshland, Trends Neurosci 6:133–137, 1983). In the article he argued that there are several similarities between neurons and bacterial cells in “how signals are processed within a cell or how this processing machinery can be modified to produce plasticity”. He then explored the bacterial chemosensory system to emphasize its attributes that are analogous to information processing in neurons. In this review, we wish to expand Koshland’s original idea by adding the yeast cell to the list of useful models of a neuron. The fact that yeasts and neurons are specialized versions of the eukaryotic cell sharing all principal components sets the stage for a grand evolutionary tinkering where these components are employed in qualitatively different tasks, but following analogous molecular logic. By way of example, we argue that evolutionarily conserved key components involved in polarization processes (from budding or mating in Saccharomyces cervisiae to neurite outgrowth or spinogenesis in neurons) are shared between yeast and neurons. This orthologous conservation of modules makes S. cervisiae an excellent model organism to investigate neurobiological questions. We substantiate this claim by providing examples of yeast models used for studying neurological diseases.

Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA Receptors

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular αx+/γ2- ( x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/β- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure-activity relationship models for benzodiazepine site activity. Considering all possible α+/β- and α+/γ- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2- site with the homologous α1+/β3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2- subunit into the β3- subunit, resulting in a quadruple mutant β3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1β3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/β- sites.

Assembly of GABAAreceptors (Review)

GABAA receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABAA receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABAA receptor subtypes in the brain. The majority of GABAA receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABAA receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABAA receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABAA receptors are discussed in the light of recent publications.