Jack A. Benson

Benzodiazepine actions mediated by specific gamma-aminobutyric acidA receptor subtypes

GABAA (γ-aminobutyric acidA) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine α1-subunit gene, that α1-type GABAA receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter γ-aminobutyric acid is preserved. α1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABAA receptors found in the limbic system (α2, α5), in monoaminergic neurons (α3) and in motoneurons (α2, α5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABAA receptor subtypes in distinct neuronal circuits, which is of interest for drug design.

Postsynaptic clustering of major GABA A receptor subtypes requires the γ2 subunit and gephyrin

Most fast inhibitory neurotransmission in the brain is mediated by GABA A receptors, which are mainly postsynaptic and consist of diverse α and ß subunits together with the γ2 subunit. Although the γ2 subunit is not necessary for receptor assembly and translocation to the cell surface, we show here that it is required for clustering of major postsynaptic GABAA receptor subtypes. Loss of GABAA receptor clusters in mice deficient in the γ2 subunit, and in cultured cortical neurons from these mice, is paralleled by loss of the synaptic clustering molecule gephyrin and synaptic GABAergic function. Conversely, inhibiting gephyrin expression causes loss of GABAA receptor clusters. The γ2 subunit and gephyrin are thus interdependent components of the same synaptic complex that is critical for postsynaptic clustering of abundant subtypes of GABAA receptors in vivo.

BDNF reduces miniature inhibitory postsynaptic currents by rapid downregulation of GABAA receptor surface expression

Changes in neurotransmitter receptor density at the synapse have been proposed as a mechanism underlying synaptic plasticity. Neurotrophic factors are known to influence synaptic strength rapidly. In the present study, we found that brain‐derived neurotrophic factor (BDNF) acts postsynaptically to reduce γ‐aminobutyric acid (GABA)‐ergic function. Using primary cultures of rat hippocampal neurons, we investigated the effects of BDNF on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and on the localization of GABAA receptors. Application of BDNF (100 ng/mL) led within minutes to a marked reduction (33.5%) of mIPSC amplitudes in 50% of neurons, recorded in the whole‐cell patch‐clamp mode, leaving frequency and decay kinetics unaffected. This effect was blocked by the protein kinase inhibitor K252a, which binds with high affinity to trkB receptors. Immunofluorescence staining with an antibody against trkB revealed that about 70% of cultured hippocampal pyramidal cells express trkB. In dual labelling experiments, use of neurobiotin injections to label the recorded cells revealed that all cells responsive to BDNF were immunopositive for trkB. Treatment of cultures with BDNF reduced the immunoreactivity for the GABAA receptor subunits‐α2, ‐β2,3 and ‐γ2 in the majority of neurons. This effect was detectable after 15 min and lasted at least 12 h. Neurotrophin‐4 (NT‐4), but not neurotrophin‐3 (NT‐3), also reduced GABAA receptor immunoreactivity, supporting the proposal that this effect is mediated by trkB. Altogether the results suggest that exposure to BDNF induces a rapid reduction in postsynaptic GABAA receptor number that is responsible for the decline in GABAergic mIPSC amplitudes.

Pharmacology of recombinant γ‐aminobutyric acidA receptors rendered diazepam‐insensitive by point‐mutated α‐subunits

Amino acids in the α‐ and γ‐subunits contribute to the benzodiazepine binding site of GABAA‐receptors. We show that the mutation of a conserved histidine residue in the N‐terminal extracellular segment (α1H101R, α2H101R, α3H126R, and α5H105R) results not only in diazepam‐insensitivity of the respective αxβ2,3γ2‐receptors but also in an increased potentiation of the GABA‐induced currents by the partial agonist bretazenil. Furthermore, Ro 15‐4513, an inverse agonist at wild‐type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABAA‐receptor subtypes in vivo.

Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification.

Ligand-gated ion channels generally display a heterooligomeric subunit structure. The present report describes an electrophysiological method that provides criteria indicating the subunit stoichiometry of a recombinant GABAA receptor composed of alpha 3, beta 2 and gamma 2 subunits. Our results exclude the stoichiometries 3 alpha 1 beta 1 gamma, 1 alpha 3 beta 1 gamma, 1 alpha 1 beta 3 gamma and suggest that the possible subunit stoichiometries for this receptor are 2 alpha 1 beta 2 gamma, 2 alpha 2 beta 1 gamma or 1 alpha 2 beta 2 gamma, of which the alpha subunit composition 2 alpha 1 beta 2 gamma may be favoured. The method is based on the quantification of the outward rectification of the GABA-evoked current induced by point mutation of charged amino acids located near the ion channel pore.

The GABAA Receptors

Transmitter-gated ion channels are multisubunit membrane-spanning receptors that serve as rapid signal transduction devices regulating the flow of cations or anions through the cell membrane. Cell type—specific flexibility in neurotransmission is accomplished by a multiplicity of channel variants based on the combinatorial assembly of structurally related subunits. The heteromeric subunit structure also permits synaptic plasticity by changes in subunit composition and subunit-specific posttranslational modification. In addition, receptor heterogeneity offers the potential for cell type-specific drug targeting.

Single-channel properties of neuronal GABAA receptors from mice lacking the γ2 subunit

1 The aim of this study was to define the biophysical properties contributed by the γ2 subunit to native single GABAA receptors. 2 Single‐channel activity was recorded from neurones of wild‐type (γ2+/+) mice and compared with that from mice which were heterozygous (γ2+/−) or homozygous (γ2−/−) for a targeted disruption in the γ2 subunit gene of the GABAA receptor. Unitary currents were evoked by low concentrations of GABA (0.5–5 μM) in membrane patches from acutely isolated dorsal root ganglion (DRG) neurones (postnatal day 0) and by 1 μM GABA in patches from embryonic hippocampal neurones which were cultured for up to 3 weeks. 3 GABAA receptors from DRG and hippocampal neurones of γ2+/+ and γ2+/− mice displayed predominantly a conductance state of 28 pS and less frequently 18 and 12 pS states. In γ2−/− mice, conductance states mainly of 12 pS and less frequently of 24 pS were found. 4 The mean open duration of the 28 pS state in γ2+/+ GABAA receptors (1.5–2.6 ms) was substantially longer than for the 12 pS state of γ2−/− GABAA receptors (0.9–1.2 ms) at all GABA concentrations. For γ2+/+ and γ2−/− channels, the mean open duration was increased at higher GABA concentrations. 5 Open duration frequency distributions of 28 and 12 pS receptors revealed the existence of at least three exponential components. Components with short mean durations declined and components with long mean durations increased in relative frequency at higher GABA concentration indicating at least two binding sites of GABA per 28 and 12 pS receptor. 6 Shut time frequency distributions revealed at least four exponential components of which two were identified as intraburst components in 28 pS and one in 12 pS GABAA receptors. 7 The mean burst duration and the mean number of openings per burst increased in 28 and 12 pS GABAA receptors with increasing GABA concentration. At least two burst types were identified: simple bursts consisting of single openings and complex bursts of five to six openings in 28 pS but only two to three openings in 12 pS GABAA receptors. 8 We conclude that the γ2 subunit enhances the efficacy of GABA by determining open conformations of high conductance and long lifetime, and by prolonging the time receptors remain in the activated bursting state.

The electrophysiological pharmacology of neurotransmitter receptors on locust neuronal somata

The mechanically-dissociated neuronal somata from the thoracic ganglia of locusts (Schistocerca gregaria and Locusta migratoria) respond in a characteristic manner to virtually all putative insect neurotransmitters, as well as to some of the few neuropeptides tested so far. The isolated somata remain eleetrophysiologically viable in vitro for many hours so that it has been possible to characterize in detail the pharmacology and ionic- and voltage-dependences of many of these responses under voltage-clamp conditions. Our current knowledge of these properties is reviewed and compared with the corresponding data available from insect synaptic responses.

GABAA-receptor assembly in vivo: Lessons from subunit mutant mice

The rules governing the assembly of GABA(A) receptors in vivo were assessed in subunit mutant mice. The transcription of individual subunit genes was regulated independently. The lack of a particular subunit did not result in a molecular rescue by an enhanced transcription of other subunits. In addition, the availability of an alpha- and beta-subunit was essential for receptor formation. Finally, highly selective recognition processes directed the subcellular targeting of receptors. The loss of a particular receptor subtype (alpha5) did not lead to a subcellular redistribution of the remaining subtype (alpha2) present in the same cell.

GABAA-Receptor Subtypes: Pharmacological Significance and Mutational Analysis in Vivo

The subunit composition of the major GABAA-receptors in vivo has been identified. They comprise about half a dozen major subtypes (e.g., αl β2γ2, α2β3γ2, α3β3γ2) and one or two dozen minor subtypes, some of which contain two α-subunit variants. The functional significance of GABAA-receptor subtypes was assessed by mutational analysis in vivo. Benzodiazepine-insensitive mice were generated by targeted disruption of the γ2-subunit gene. For drug development, GABAA-receptor heterogeneity offers new opportunities to advance the therapy of anxiety, insomnia, and epilepsy with ligands acting at selected modulatory sites.