Kavain, the Major Constituent of the Anxiolytic Kava Extract, Potentiates GABAA Receptors: Functional Characteristics and Molecular Mechanism.

Extracts of the pepper plant kava (Piper methysticum) are effective in alleviating anxiety in clinical trials. Despite the long-standing therapeutic interest in kava, the molecular target(s) of the pharmacologically active constituents, kavalactones have not been established. γ-Aminobutyric acid type A receptors (GABAARs) are assumed to be the in vivo molecular target of kavalactones based on data from binding assays, but evidence in support of a direct interaction between kavalactones and GABAARs is scarce and equivocal. In this study, we characterised the functional properties of the major anxiolytic kavalactone, kavain at human recombinant α1ß2, ß2γ2L, αxß2γ2L (x = 1, 2, 3 and 5), α1ßxγ2L (x = 1, 2 and 3) and α4ß2δ GABAARs expressed in Xenopus oocytes using the two-electrode voltage clamp technique. We found that kavain positively modulated all receptors regardless of the subunit composition, but the degree of enhancement was greater at α4ß2δ than at α1ß2γ2L GABAARs. The modulatory effect of kavain was unaffected by flumazenil, indicating that kavain did not enhance GABAARs via the classical benzodiazepine binding site. The ß3N265M point mutation which has been previously shown to profoundly decrease anaesthetic sensitivity, also diminished kavain-mediated potentiation. To our knowledge, this study is the first report of the functional characteristics of a single kavalactone at distinct GABAAR subtypes, and presents the first experimental evidence in support of a direct interaction between a kavalactone and GABAARs.

A Multifaceted GABAA Receptor Modulator: Functional Properties and Mechanism of Action of the Sedative-Hypnotic and Recreational Drug Methaqualone (Quaalude).

In the present study, we have elucidated the functional characteristics and mechanism of action of methaqualone (2-methyl-3-o-tolyl-4(3H)-quinazolinone, Quaalude), an infamous sedative-hypnotic and recreational drug from the 1960s-1970s. Methaqualone was demonstrated to be a positive allosteric modulator at human α1,2,3,5ß2,3γ2S GABAA receptors (GABAARs) expressed in Xenopus oocytes, whereas it displayed highly diverse functionalities at the α4,6ß1,2,3δ GABAAR subtypes, ranging from inactivity (α4ß1δ), through negative (α6ß1δ) or positive allosteric modulation (α4ß2δ, α6ß2,3δ), to superagonism (α4ß3δ). Methaqualone did not interact with the benzodiazepine, barbiturate, or neurosteroid binding sites in the GABAAR. Instead, the compound is proposed to act through the transmembrane ß((+))/α((-)) subunit interface of the receptor, possibly targeting a site overlapping with that of the general anesthetic etomidate. The negligible activities displayed by methaqualone at numerous neurotransmitter receptors and transporters in an elaborate screening for additional putative central nervous system (CNS) targets suggest that it is a selective GABAAR modulator. The mode of action of methaqualone was further investigated in multichannel recordings from primary frontal cortex networks, where the overall activity changes induced by the compound at 1-100 µM concentrations were quite similar to those mediated by other CNS depressants. Finally, the free methaqualone concentrations in the mouse brain arising from doses producing significant in vivo effects in assays for locomotion and anticonvulsant activity correlated fairly well with its potencies as a modulator at the recombinant GABAARs. Hence, we propose that the multifaceted functional properties exhibited by methaqualone at GABAARs give rise to its effects as a therapeutic and recreational drug.

Probing α4ßδ GABAA receptor heterogeneity: differential regional effects of a functionally selective α4ß1δ/α4ß3δ receptor agonist on tonic and phasic inhibition in rat brain.

In the present study, the orthosteric GABAA receptor (GABAAR) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABAARs and native rat GABAARs. Whereas Thio-THIP displayed weak antagonist activity at α1,2,5ß2,3γ2S and ρ1 GABAARs and partial agonism at α6ß2,3δ GABAARs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at α4ß1δ and α4ß3δ GABAARs was contrasted by its negligible activity at the α4ß2δ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABAARs, we assessed the effects of 100 µm Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its α6ß2,3δ agonism, Thio-THIP evoked robust currents through extrasynaptic GABAARs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4ß2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic α4ßδ subtypes in the cells. Finally, whereas 100 µm Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4ßδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ß2- and ß3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native α4ßδ GABAARs.

Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4ßδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4ß3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

Pharmacological characterisation of α6ß4⁎ nicotinic acetylcholine receptors assembled from three chimeric α6/α3 subunits in tsA201 cells.

The distribution and physiological functions of the α6 subunit-containing (α6⁎) nicotinic acetylcholine receptors in the central nervous system make them interesting putative therapeutic targets in several disorders. However, investigations into the receptors have been complicated by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6ß4 and α6ß4ß3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L) identified in a recent study (Jensen et al., 2013). Whereas the Bmax values exhibited by [3H]epibatidine at wild-type α6ß4, C1ß4, C6F223Lß4 and C16F223Lß4 receptors differed substantially, the radioligand and seven orthosteric nicotinic agonists exhibited very similar KD and Ki values at the four receptors. In the FLIPR™ Membrane Potential Blue assay, the agonists exhibited the same rank order of potencies [(±)-epibatidine>sazetidine A>varenicline>(-)-cytisine~(S)-nicotine>acetylcholine>carbachol] at the C1ß4, C1ß4ß3, C6F223Lß4, C6F223Lß4ß3, C16F223Lß4 and C16F223Lß4ß3 receptors, albeit the absolute EC50 values differed somewhat between the receptors. Furthermore, four reference antagonists displayed the same rank order of inhibitory potencies at the six receptors [α-conotoxin PIA>2,2,6,6-tetramethylpiperidin-4-yl heptanoate>mecamylamine>dihydro-ß-erythroidine]. Although all interpretations based on these results should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6ß4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors.

Elucidation of molecular impediments in the α6 subunit for in vitro expression of functional α6ß4* nicotinic acetylcholine receptors.

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with ß4 or ß4ß3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6ß4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6ß4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6ß4ß3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with ß2 or ß2ß3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6ß4* nAChRs.

Positive modulation of δ-subunit containing GABA(A) receptors in mouse neurons.

δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 µM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 µM), while interneurons showed a potentiation of 2.6-fold. Moreover, AA29504 (1 µM) increased the amplitude and prolonged the decay of miniature inhibitory postsynaptic currents (mIPSCs) in granule cells, and this effect was abolished by Zn²âº (15 µM). AA29504 (1 µM) also induced a small tonic current (12.7 ± 3.2 pA) per se, and when evaluated in a nominally GABA-free environment using Ca²âº imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition, and possibly recruits perisynaptic δ-containing receptors to participate in synaptic phasic inhibition in dentate gyrus.

Influence of the Chlamydia pneumoniae AR39 bacteriophage ϕCPAR39 on chlamydial inclusion morphology.

The human respiratory tract pathogen Chlamydia pneumoniae AR39 is naturally infected by the bacteriophage ϕCPAR39. The phage genome encodes six ORFs, [ORF8, ORF4, ORF5, and viral protein (VP) 1, VP2 and VP3]. To study the growth of the phage, antibodies were generated to VP1 and used to investigate the ϕCPAR39 infection. Using immunofluorescence laser confocal microscopy and two-dimensional gel electrophoresis, we investigated the ϕCPAR39 infection of C. pneumoniae AR39. It was observed that ϕCPAR39 infection differentially suppressed the C. pneumoniae protein synthesis as the polymorphic membrane protein 10 and the secreted chlamydial protein Cpn0796 was hardly expressed while the secreted chlamydial protein Cpaf was expressed, but not secreted. The inclusion membrane protein, IncA, was demonstrated to surround the phage-infected abnormal reticulate bodies (RB) as well as being located in the inclusion membrane. As IncA is secreted by the type 3 secretion (T3S) system, it is likely that the T3S is disrupted in the phage-infected chlamydiae such that it accumulates around the infected RB.

Modulation of extrasynaptic THIP conductances by GABAA-receptor modulators in mouse neocortex.

THIP is a hypnotic drug, which displays a unique pharmacological profile, because it activates a subset of extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors containing delta-subunits. It is important to study the physiology and pharmacology of these extrasynaptic receptors and to determine how THIP interacts with other hypnotics and anesthetics. Here, we study the modulation of the extrasynaptic response to THIP using three classes of GABA(A)-receptor ligands. In whole cell recordings from mouse neocortical layer 2/3 pyramidal cells, THIP induced an extrasynaptic tonic current of 44 +/- 5 pA. The benzodiazepine site agonist and hypnotic zolpidem (500 nM), which displays selectivity for alpha(1/2/3)- and gamma(2)-containing receptors, did not alter the tonic current induced by THIP. The anesthetic etomidate (1 microM), which shows selectivity for beta(2)- and beta(3)-containing GABA(A) receptors, potentiated the THIP current by 126%. Etomidate also induced a small tonic GABA(A) current per se. The anesthetic propofol (1 microM), which displays broad-spectrum modulatory effects on several GABA(A)-receptor subtypes, enhanced the tonic THIP current by 117%. Finally, all three compounds modulated the function of intrasynaptic receptors activated by synaptically released GABA. Our study shows that the extrasynaptic GABA(A) receptors responsible for the tonic THIP conductance likely do not contain alpha(1)-, alpha(2)-, alpha(3)-, and gamma(2)-subunits. Thus the tonic GABAergic conductance in the neocortex is presumably mediated by alpha(4)beta(2/3)delta receptors, which are likely to play a major role for neocortical excitability. Furthermore, our study has deepened the knowledge about the cellular actions of THIP as well as THIP's interactions with other hypnotics and anesthetics.