Roland Baur

The effect of subunit composition of rat brain GABAA receptors on channel function

Different combinations of cloned rat brain subunit isoforms of the GABAA receptor channel were expressed in Xenopus oocytes. The voltage-clamp technique was then used to measure properties of the GABA-induced membrane currents and to study the effects of various modulators of the GABAA receptor channel (diazepam, DMCM, pentobarbital, and picrotoxin). This approach was used to obtain information on the minimal structural requirements for several functional properties of the ion channel. The combination alpha 5 beta 2 gamma 2 was identified as the minimal requirement reproducing consensus properties of the vertebrate GABAA receptor channel, including cooperativity of GABA-dependent channel gating with a Ka in the range of 10 microM, modulation by various drugs acting at the benzodiazepine binding site, picrotoxin sensitivity, and barbiturate effects.

Anxiolytic Effect of Wogonin, a Benzodiazepine Receptor Ligand Isolated from Scutellaria Baicalensis Georgi

The search for novel anxiolytics devoid of undesirable side-effects typical of classical benzodiazepines (BDZs) has been intense, and flavonoids, as a relative new class of ligands, have been shown to possess anxiolytic effects in vivo. The present study evaluated the pharmacological properties of a naturally occurring monoflavonoid, 5,7-dihydroxy-8-methoxyflavone or wogonin. The affinity (K(i)) of wogonin for the benzodiazepine site (BZD-S) on the gamma-aminobutyric acid(A) (GABA(A)) receptor complex was 0.92 microM. Using electrophysiological techniques, we showed that wogonin enhanced the GABA-activated current in rat dorsal root ganglion neurons, and in Xenopus laevis oocytes expressing recombinant rat GABA(A) receptors, the enhancement was partially reversed by the co-application of a 1 microM concentration of the BZD-S antagonist anexate (Ro15-1788). Acute toxicity and behavioral effects were examined in mice. Acute lethal activity was low, with an LD(50) of 3.9 g/kg. Oral administration of wogonin (7.5 to 30 mg/kg) elicited an anxiolytic response that was similar to that elicited by diazepam in the elevated plus-maze; a dose-dependent increase in open arm entries and time spent in open arms was observed. More importantly, its anxiolytic effect was blocked by the co-administration of Ro15-1788. In the holeboard test, not only did wogonin-treated mice experience an increased number of head-dips but they also spent more time at it, showing no signs of sedation. Furthermore, wogonin did not cause myorelaxant effects in the horizontal wire test. Taken together, these data suggest that wogonin exerts its anxiolytic effect through positive allosteric modulation of the GABA(A) receptor complex via interaction at the BZD-S. Its anxiolytic effect was not accompanied by sedative and myorelaxant side-effects typical of BDZs.

The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood–brain barrier and exhibits anticonvulsive effects

The functional characterization of hispidulin (4′,5,7‐trihydroxy‐6‐methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABAA/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nM and higher stimulated the GABA‐induced chloride currents at tested receptor subtypes (α1−3,5,6β2γ2S) indicating positive allosteric properties. Maximal stimulation at α1β2γ2S was observed with 10 μM hispidulin. In contrast to diazepam, hispidulin modulated the α6β2γ2S‐GABAA receptor subtype. When fed to seizure‐prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg−1 body weight (BW) per day) and diazepam (2 mg kg−1 BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood–brain barrier for the chemically synthesized, 14C‐labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (Kin) of 1.14 ml min−1 g−1, measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco‐2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 μM, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites.

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABAA receptors in neuronal populations within the olfactory bulb.

Recombinant GABAA receptor function and ethanol.

Different combinations of cloned subunits of the rat brain GABAA receptor were expressed in Xenopus oocytes. Possible effects of ethanol on the expressed GABA‐induced chloride current were determined. The consequence of replacing the γ2s subunit by the alternatively spliced variant γ2L was specifically tested on the responsiveness to ethanol. A significant stimulation of the GABA response was only observed at very high concentrations (> 60 mM) of ethanol. No differential response was observed between subunit combinations containing different γ2 subunit splice variants.

The relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAA receptors expressed in Xenopus oocytes

Benzodiazepine (BZD) potentiation of GABA-activated Cl(-)-current (I(GABA)) in recombinant GABA(A) receptors requires the presence of the gamma subunit. When alpha1, beta2 and gamma2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of I(GABA) is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the gamma2S subunit. In addition, GABA EC(50) values for alpha1beta2gamma2 (1:1:1) receptors are intermediate to values measured for alpha1beta2 (1:1) and alpha1beta2gamma2 (1:1:10) receptors. We conclude that co-expression of equal ratios of alpha1, beta2 and gamma2 subunits in Xenopus oocytes produces a mixed population of alpha1beta2 and alpha1beta2gamma2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of gamma2 subunit cRNA relative to alpha1 and beta2 cRNA. This results in a purer population of alpha1beta2gamma2 receptors.

Activation of protein kinase C results in down-modulation of different recombinant GABAA-channels.

Different combinations of cloned rat brain subunits of the GABAA receptor were expressed in Xenopus oocytes. The effect of the phorbol ester PMA, an activator of protein kinase C, on the expressed GABA‐gated ion current was determined. Ion currents were diminished by β‐PMA, but not by the control substance α‐PMA, irrespective of the subunit combination studied. The mechanism of current decrease was investigated in more detail for the subunit combination α5β2γ2. The reversal potential of the current remained unaffected, while the maximal current amplitude was decreased and the apparent K a for GABA‐dependent channel gating was shifted to higher concentrations.

A GABAA receptor of defined subunit composition and positioning : Concatenation of five subunits

We show that the five subunits of a γ‐aminobutyric acid type A receptor (GABAA receptor) can be concatenated to yield a functional receptor. This concatenated receptor α1–β2–α1–γ2–β2 has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non‐concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non‐concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABAA receptor subtype selective compounds.

The major central endocannabinoid directly acts at GABA(A) receptors.

GABAA receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABAA receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β2 confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit–containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB1/CB2 cannabinoid receptor double-KO, whereas β2-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABAA receptor may have far-reaching consequences for the study of locomotion and sedation.

The rat β1 ‐subunit of the GABAA receptor forms a picrotoxin‐sensitive anion channel open in the absence of GABA

The structural basis of GABA‐gated chloride channels in mammalian brain is presently explored by the functonal expression of cDNAs coding for the α, β or γ‐subunits of the receptor and their isoforms. In this context, we expressed the cloned cDNA coding for the rat β1‐subunit of the GABAA receptor in the Xenopus oocyte. Surprisingly, efficient expression of a functional ion channel was found. The channel was anion‐selective, and able to open in the absence of GABA. Since this channel could be shut by the GABA‐channel blocker picrotoxin, we conclude that the β1 ‐subunit of the GABAA receptor is sufficient to form binding sites for picrotoxin.