Hartmut Lüddens

THE DIVERSITY OF GABAA RECEPTORS : PHARMACOLOGICAL AND ELECTROPHYSIOLOGICAL PROPERTIES OF GABAA CHANNEL SUBTYPES

The amino acid γ-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotrans-mitter that mediates most of its effects through fast GABA-gated Cl−-channels (GABAAR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes α, β, γ, δ, ε, and ρ, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABAARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and ethanol. To understand the physiology and diversity of GABAARs, the native isoforms have to be identified by their localization in the brain and by their pharmacology. In heterologous expression systems, channels require the presence of α, β, and γ subunits in order to mimic the full repertoire of native receptor responses to drugs, with the BZ pharmacology being determined by the particular α and γ subunit variants. Little is known about the functional properties of the β, δ, and ε subunit classes and only a few receptor subtype-specific substances like loreclezole and furosemide are known that enable the identification of defined receptor subtypes. We will summarize the pharmacology of putative receptor isoforms and emphasize the characteristics of functional channels. Knowledge of the complex pharmacology of GABAARs might eventually enable site-directed drug design to further our understanding of GABA-related disorders and of the complex interaction of excitatory and inhibitory mechanisms in neuronal processing.

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor α4 subunit

A cDNA of rat brain encoding the GABAA receptor α4 subunit has been cloned. Recombinant receptors composed of α4, β2 and γ2 subunits bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine ‘alcohol antagonist’ [3H]Ro 15‐4513, but fail to bind benzodiazepine agonists. The α4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The α4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.

NMDA receptor channels: Subunit-specific potentiation by reducing agents

Sulfhydryl redox agents affect NMDA receptor activity. We investigated a putative redox site in four recombinant NMDA receptors. In 293 cells expressing NR1-NR2A channels dithiothreitol (DTT) rapidly potentiated L-glutamate-activated whole-cell currents and decreased the time course of desensitization and deactivation. Part of the current potentiation (reversible component) and all kinetic changes reversed upon washout. The remaining potentiation (persistent component) was abolished by an oxidizing agent. The N-terminal 370 residues of NR2A mediate the reversible component in chimeric NR2 subunits. In cells expressing the NR1-NR2B, -NR2C, and -NR2D channels DTT elicited only a slowly developing, persistent potentiation and increased the deactivation time course. In these, but not in NR1-NR2A, the DTT effect was rendered insensitive to reoxidation by alkylation. Reduced glutathione mimicked the DTT effects only in the NR1-NR2A receptor. Hence, molecularly distinct NMDA receptors differ profoundly in their responses to sulfhydryl redox agents.

Oxytocin Regulates Neurosteroid Modulation of GABA A Receptors in Supraoptic Nucleus around Parturition

In this study, we investigate how neurosteroid sensitivity of GABAA receptors (GABAARs) is regulated. We examined this issue in neurons of the supraoptic nucleus (SON) of the rat and found that, during parturition, the GABAARs become insensitive to the neurosteroid allopregnanolone attributable to a shift in the balance between the activities of endogenous Ser/Thr phosphatase and PKC. In particular, a constitutive endogenous tone of oxytocin within the SON after parturition suppressed neurosteroid sensitivity of GABAARs via activation of PKC. Vice versa before parturition, during late pregnancy, application of exogenous oxytocin brings the GABAARs from a neurosteroid-sensitive mode toward a condition in which the receptors are not sensitive. This indicates that there may be an inverse causal relationship between the extent to which the GABAAR or one of its interacting proteins is phosphorylated and the neurosteroid sensitivity of the GABAAR. Neurosteroid sensitivity was not affected by changes in subunit composition of GABAARs known to occur concurrently in these cells.

Altered receptor subtypes in the forebrain of GABAA receptor δ subunit-deficient mice: recruitment of γ2 subunits

Abstract A GABAA receptor δ subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABAA receptors. High-affinity [3H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of δ−/− animals. [3H]Ro 15-4513 binding to benzodiazepine sites was increased in δ−/− animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of γ2 subunits with α4 subunits. In the western blots of forebrain membranes of δ−/− animals, the level of γ2 subunit was increased and that of α4 decreased, while the level of α1 subunits remained unchanged. In the δ−/− forebrains, the remaining α4 subunits were associated more often with γ2 subunits, since there was an increase in the α4 subunit level immunoprecipitated by the γ2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[35S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of α4γ2 and α6γ2 subunit-containing receptors, respectively. The altered pharmacology of forebrain GABAA receptors and the decrease of the α4 subunit level in δ subunit-deficient mice suggest that the δ subunit preferentially assembles with the α4 subunit. The δ subunit seems to interfere with the co-assembly of α4 and γ2 subunits and, therefore, in its absence, the γ2 subunit is recruited into a larger population of α4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABAA receptors.

Expression patterns of GABAA receptor subtypes in developing hippocampal neurons.

Abstract Developing cultured hippocampal neurons were studied for the expression of GABA A receptor subunits using anti-peptide antibodies. The antibodies decorated the plasma membrane of cell bodies and all neurites in immature neurons, whereas the immunoreactivity was restricted to somata and dendrites in fully developed cells. Most receptor subunits were expressed throughout the entire culture period. However, the α1 and γ2 subunits were detected only late in culture, while α6 disappeared around this time. These findings were partly corroborated by in situ hybridization data in the developing rat hippocampus. Our results reveal a temporal control of gene expression for subsets of receptor subunits in hippocampal neurons and suggest a change in the subunit composition of GABA A receptors in the developing brain.

Biological function of GABAA/benzodiazepine receptor heterogeneity

gamma-Aminobutyric acid (GABA) is the most prominent of the inhibiting neurotransmitters in the brain. It exerts its main action through GABAA receptors. The receptors respond to the presence of GABA by the opening of an intrinsic anion channel. Hence, they belong to the molecular superfamily of ligand-gated ion channels. There exist in the brain multiple GABAA receptors that show differential distribution and developmental patterns. The receptors presumably form by the assembly of five proteins from at least three different subunits (alpha 1-6, beta 1-3 and gamma 1-3). The regulation of functional properties by benzodiazepine (BZ) receptor ligands, neurosteroids, GABA and its analogs differs dramatically with the alpha variant present in the complex. Additional variation of the GABAA receptors comes with the exchange of the gamma subunits. No clear picture exists for the role of the beta subunits, though they may play an important part in the sensitivity of the channel-receptor complex. The effects of BZ receptor ligands on animal behavior range from agonist effects, e.g. anxiolysis, sedation, and hypnosis, to inverse agonist effects, e.g. anxiety, alertness, and convulsions. The diversity of effects reflects the ubiquity of the GABAA/BZ receptors in the brain. Recent data provide some insight into the mechanism of action of BZ ligands, but no clear delineation can be drawn from a single ligand to a single behavioral effect. This may be due to the fact that intrinsic efficacies of the ligands differ between receptor subtypes, so that the diversity of native receptors is further complicated by the diversity of the mode the ligands act on GABAA receptor subtypes. The behavioral actions of alcohol (ethanol) are similar to those produced by GABAA receptor agonists. In agreement, alcohol-induced potentiation of GABAergic responses has often been observed at behavioral, electrophysiological and biochemical levels. Thus, there is clearly a GABAA-dependent component in the actions of alcohol. However, the site and mode of action of ethanol on GABAA/BZ receptors remain controversial.

Ketamine, But Not Phencyclidine, Selectively Modulates Cerebellar GABAA Receptors Containing α6 and δ Subunits

Phencyclidine (PCP) and ketamine are dissociative anesthetics capable of inducing analgesia, psychomimetic behavior, and a catatonic state of unconsciousness. Despite broad similarities, there are notable differences between the clinical actions of ketamine and PCP. Ketamine has a lower incidence of adverse effects and generally produces greater CNS depression than PCP. Both noncompetitively inhibit NMDA receptors, yet there is little evidence that these drugs affect GABAA receptors, the primary target of most anesthetics. α6β2/3δ receptors are subtypes of the GABAA receptor family and are abundantly expressed in granular neurons within the adult cerebellum. Here, using an oocyte expression system, we show that at anesthetically relevant concentrations, ketamine, but not PCP, modulates α6β2δ and α6β3δ receptors. Additionally, at higher concentrations, ketamine directly activates these GABAA receptors. Comparatively, dizocilpine (MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]), a potent noncompetitive antagonist of NMDA receptors that is structurally unrelated to PCP, did not produce any effect on α6β2δ receptors. Of the recombinant GABAA receptor subtypes examined (α1β2, α1β2γ2, α1β2δ, α4β2γ2, α4β2δ, α6β2γ2, α6β2δ, and α6β3δ), the actions of ketamine were unique to α6β2δ and α6β3δ receptors. In dissociated granule neurons and cerebellar slice recordings, ketamine potentiated the GABAergic conductance arising from α6-containing GABAA receptors, whereas PCP showed no effect. Furthermore, ketamine potentiation was absent in cerebellar granule neurons from transgenic functionally null α6−/− and δ−/−mice. These findings suggest that the higher CNS depressant level achieved by ketamine may be the result of its selective actions on α6β2/3δ receptors.

A53T-Alpha-Synuclein Overexpression Impairs Dopamine Signaling and Striatal Synaptic Plasticity in Old Mice

Background Parkinsons disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Methodology/Principal Findings Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice. Conclusions/Significance Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches.

GABAA-receptor Subtypes: Clinical Efficacy and Selectivity of Benzodiazepine Site Ligands

The main inhibitory neurotransmitter receptor of the brain, the gamma-aminobutyric acid type A receptor (GABA[A]), mediates the actions of several classes of clinically important drugs, such as benzodiazepines, barbiturates and general anaesthetics. This review summarizes the current knowledge on how classical benzodiazepines and novel nonbenzodiazepine compounds act on the benzodiazepine site of GABA(A) receptors and on their clinical pharmacology related to anxiolytic, sedative, hypnotic and cognitive effects or side-effects. Partial agonism, receptor subtype selectivity and novel binding sites are discussed as possible strategies to develop new drugs with fewer adverse effects than are seen in the clinical use of benzodiazepines.