GABA(A) (gamma-aminobutyric acid type A) receptors mediate most of the 'fast' synaptic inhibition in the mammalian brain and are targeted by many clinically important drugs. Certain naturally occurring pregnane steroids can potently and specifically enhance GABA(A) receptor function in a nongenomic (direct) manner, and consequently have anxiolytic, analgesic, anticonvulsant, sedative, hypnotic and anaesthetic properties. These steroids not only act as remote endocrine messengers, but also can be synthesized in the brain, where they modify neuronal activity locally by modulating GABA(A) receptor function. Such 'neurosteroids' can influence mood and behaviour in various physiological and pathophysiological situations, and might contribute to the behavioural effects of psychoactive drugs.

The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT3 receptors). Recombinant expression of the only identified receptor subunit (5-HT3A) yields functional 5-HT3 receptors. However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT3 receptors (9-17 pS). Neuronal 5-HT3 receptors also display a permeability to calcium ions and a current-voltage relationship that differ from those of homomeric receptors. Here we describe a new class of 5-HT3-receptor subunit (5-HT3B). Transcripts of this subunit are co-expressed with the 5-HT3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT3A and 5-HT3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current-voltage relationship which resembles that of characterized neuronal 5-HT3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

The principal inhibitory neurotransmitter in the mammalian brain, ␥-aminobutyric acid (GABA), is thought to regulate memory processes by activating transient inhibitory postsynaptic currents. Here we describe a nonsynaptic, tonic form of inhibition in mouse CA1 pyramidal neurons that is generated by a distinct subpopulation of GABA type A receptors (GABA ARs). This tonic inhibitory conductance is predominantly mediated by ␣5 subunit-containing GABAARs (␣5GABAARs) that have different pharmacological and kinetic properties compared to postsynaptic receptors. GABAARs that mediate the tonic conductance are well suited to detect low, persistent, ambient concentrations of GABA in the extracellular space because they are highly sensitive to GABA and desensitize slowly. Moreover, the tonic current is highly sensitive to enhancement by amnestic drugs. Given the restricted expression of ␣5GABAARs to the hippocampus and the association between reduced ␣5GABAAR function and improved memory performance in behavioral studies, our results suggest that tonic inhibition mediated by ␣5GABAARs in hippocampal pyramidal neurons plays a key role in cognitive processes. The ␥-aminobutyric acid (GABA) subtype A receptor (GABA A R) is a pentameric anion-selective ion channel that assembles from different classes of subunits (␣1-6, ␤1-3, ␥1-3, ␦, , , and ) (1). The combination of various GABA A R subunits confers different biophysical and pharmacological properties and regulates regional and subcellular patterns of distribution (2, 3). The subunit composition critically determines agonist affinity, receptor kinetics, and sensitivity to a variety of clinically important drugs, including benzodiazepines and general anesthetics.Studies of gene-targeted mice have implicated specific GABA A R subunit isoforms in critical aspects of information processing in the brain. Notably, ␣5-null mutant mice (␣5Ϫ͞Ϫ) exhibit improved performance in the water maze model of spatial learning, a hippocampus-dependent learning task (4). Further, mice carrying a point mutation at position 105 of the ␣5 subunit (H105R) experience an unexpected selective reduction of ␣5GABA A Rs in hippocampal pyramidal neurons and improved performance for learning tasks (5). Pharmacological studies further support the involvement of ␣5GABA A Rs in learning processes; for example, ␣5 subunit-selective inverse agonists such as L-655,708 enhance learning performance in rats in the Morris water maze test (6, 7). Moreover, ␣5 subunitselective inverse agonists exhibit desirable nootropic effects without causing adverse convulsant activities associated with nonselective GABA A R inverse agonists. Thus, inhibition of ␣5GABA A Rs presents an attractive strategy for developing memory-enhancing drugs.The neuronal substrates underlying improved cognitive performance associated with reduced ␣5GABA A R function remain unknown. The ␣5 subunit has a unique and limited pattern of distribution in the mammalian brain. Although ␣5-containing receptors constitute Ͻ5% of the total GABA A R population...

No abstract

The ␥-aminobutyric acid type A (GABA A ) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦ 1 , and 1 ). Positive allosteric modulation of GABA A receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABA A receptors is uniquely dependent upon the ␤ subunit subtype present within the receptor. Receptors containing ␤ 2 -or ␤ 3 -, but not ␤ 1 subunits, are highly sensitive to the agent. Here, chimeric ␤ 1 ͞␤ 2 subunits coexpressed in Xenopus laevis oocytes with human ␣ 6 and ␥ 2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the ␤ 3 subunit to Ser (the homologous residue in ␤ 1 ), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the ␤ 1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk؊) cells stably expressing the ␣ 6 ␤ 3 ␥ 2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

No abstract

Among hypnotic agents that enhance GABA A receptor function, etomidate is unusual because it is selective for ␤ 2 /␤ 3 compared with ␤ 1 subunit-containing GABA A receptors. Mice incorporating an etomidate-insensitive ␤ 2 subunit (␤ 2N265S ) revealed that ␤ 2 subunitcontaining receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the ␤ 2 -containing receptors that mediate these actions is unknown, the thalamus is implicated.We have characterized GABA A receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, ␤ 2 Ϫ/Ϫ , and ␤ 2N265S mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes ϳ80% of the total GABA A receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the ␤ 2N265S mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nM to 3 M) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.