Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and ...analyzed two mouse lines in which the α2 or α3 GABAA(γ-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the α2(H101R) point mutation but present in mice with the α3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by α2 GABAAreceptors, which are largely expressed in the limbic system, but not by α3 GABAAreceptors, which predominate in the reticular activating system.
Main-group metals are inherently labile, hindering their use in catalysis. We exploit this lability in the synthesis of isocyanurates. For the first time we report a highly active catalyst that ...trimerizes alkyl, allyl and aryl isocyanates, and di-isocyanates, with low catalyst loadings under mild conditions, using a hemi-labile aluminium-pyridyl-bis(iminophenolate) complex.
The lability of aluminium is exploited to give a highly effective catalyst for the trimerization of isocyanates.
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
Synaptic localization of γ-aminobutyric acid type A (GABA A ) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to ...postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABA A receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABA A receptors that are synaptically clustered and colocalized with gephyrin in vivo . These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABA A receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABA A receptors in wild-type mice.
ABSTRACT
Mechanically isolated neuronal somata from the thoracic ganglia of the locust Locusta migratoria remain electrophysiologically viable under current-or voltage-clamp in vitro for many hours. ...Nicotine and muscarine evoke different responses when pressure-microapplied to these somata. The response to acetylcholine is mainly nicotinic but contains a small muscarinic component. The nicotinic (AChl) response is a rapid depolarisation accompanied by a decrease in membrane resistance. In voltage-clamped somata, the current mediating the AChl response is inward over the membrane potential range −30 to − 110 mV, decreasing with depolarisation and with a projected reversal potential of about +20 mV. The muscarinic (ACh2) response is a slow depolarisation accompanied by a decrease in membrane resistance. In voltage-clamped somata, the current mediating the ACh2 response is inward, decreasing to zero at potentials of −80 to −90 mV. The AChl response is evoked by nicotine, anabasine, tetramethylammonium, DMPP and relatively high concentrations of the nitromethylene heterocycle insecticide, PMNI. Suberyldicholine or decamethonium evoke the response only when acetylcholine is present in the bathing saline. Nicotinic antagonists of the AChl response, in descending order of potency, are PMN1 > α-bungarotoxin⩾lobeline⩾mecamylamine>trimethaphan camsylate>chlorisondamine⩾d-tubo-curarine⩾hexamethomium⩾gallamine triethiodide⩾tetracthylammonium. This response is also potently blocked by strychnine and more weakly blocked by δ-philanthotoxin, bicuculline and picrotoxin. The ACh2 response is evoked by muscarine, oxotremorine, arecoline, pilocarpine and, very weakly, by the Mj-selective agonist McN-A-343. Muscarinic antagonists of the ACh2 response, in descending order of potency, are QNB> scopolamine>atropine>4-DAMP (M3) ⩾benactyzine⩾HHSiD (M1/M3) ⩾ pirenzepine (M1). QNX (M1), AF-DX116 (M2), gallamine triethiodide (M2) and methoctramine (M2) are almost or completely inactive. With the exception of pirenzepine and QNX, all of the muscarinic antagonists used in this study also block the nicotinic AChl response with EC50 values in the range 5 to 50μmol l−1, similar to those for δ-philanthotoxin, bicuculline and picrotoxin. Pirenzepine is inactive (10μmol l−1), but QNX is potently active, with an EC50 value of approximately 20 nmol l−1, similar to that of α-bungarotoxin. The extrasynaptic nicotinic and muscarinic receptors of Locusta migratoria neurones are pharmacologically distinct from the corresponding mammalian receptors studied so far.
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.
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