Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences.

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism.

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ∼10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est) and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louse's simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development.

Exclusion of DMRT1 as a candidate gene for canine SRY-negative XX sex reversal.

DMRT1, which encodes a zinc finger-like DNA binding motif, is a well-conserved gene that is involved in testis differentiation in a variety of mammalian and non-mammalian vertebrates. The objective of this study was to determine whether a DMRT1 microsatellite marker allele is associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal generated from an American Cocker spaniel founder. Ten affected dogs and their parents and grandparents were genotyped. Four alleles at this locus and five different genotypes were found in this pedigree. All affected dogs inherited this trait from the foundation sire of this colony. Thus, the disease-causing mutation should be identical by descent in all affected dogs. Six affected dogs were found to have genotypes at this locus that were different from those of the founder sire. These results indicate that DMRT1 is an unlikely candidate gene for SRY-negative XX sex reversal in this model.

A marker set for construction of a genetic map of the silver fox (Vulpes vulpes).

The silver fox, a variant of the red fox (Vulpes vulpes), is a close relative of the dog (Canis familiaris). Cytogenetic differences and similarities between these species are well understood, but their genomic organizations have not been compared at higher resolution. Differences in their behavior also remain unexplained. Two silver fox strains demonstrating markedly different behavior have been generated at the Institute of Cytology and Genetics of the Russian Academy of Sciences. Foxes selected for tameness are friendly, like domestic dogs, while foxes selected for aggression resist human contact. To refine our understanding of the comparative genomic organization of dogs and foxes, and enable a study of the genetic basis of behavior in these fox strains, we need a meiotic linkage map of the fox. Towards this goal we generated a primary set of fox microsatellite markers. Four hundred canine microsatellites, evenly distributed throughout the canine genome, have been identified that amplify robustly from fox DNA. Polymorphism information content (PIC) values were calculated for a representative subset of these markers and population inbreeding coefficients were determined for tame and aggressive foxes. To begin to identify fox-specific single nucleotide polymorphisms (SNPs) in genes involved in the neurobiology of behavior, fox and dog orthologs of serotonin 5-HT1A and 5-HT1B receptor genes have been cloned. Sequence comparison of these genes from tame and aggressive foxes reveal several SNPs. The close relationship of the fox and dog enables canine genomic tools to be utilized in developing a fox meiotic map and mapping behavioral traits in the fox.

The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance.

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, out with the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the 'HA stretch') within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.

Introduction of the 5-HT3B subunit alters the functional properties of 5-HT3 receptors native to neuroblastoma cells.

The identification of a second 5-HT(3) (5-HT(3B)) subunit provides an explanation for 5-HT(3) receptor heterogeneity. We investigated whether introduction of recombinant 5-HT(3B) subunits would alter the functional properties of mouse neuroblastoma 5-HT(3) receptors. RT-PCR analysis revealed that NB41A3 cells contain mRNAs encoding 5-HT(3A) and 5-HT(3B) subunits. 5-HT increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and caused the concentration-dependent activation of inward currents recorded at -60 mV. Both actions of 5-HT were antagonized by ondansetron. The 5-HT concentration-response relationship of NB41A3 cells was indistinguishable from that of the related NG108-15 cell line. The selective 5-HT(3)-receptor agonist mCPBG also elevated [Ca(2+)](i) and activated inward currents. 2-M-5HT was less efficacious than 5-HT as an activator of 5-HT(3) receptors in NB41A3 cells and did not significantly increase [Ca(2+)](i). The 5-HT induced increase in [Ca(2+)](i) did not involve caffeine- or thapsigargin-sensitive intracellular Ca(2+) stores. The introduction of the 5-HT(3B) subunit by transient transfection of NB41A3 cells caused 5-HT to become less potent as an activator of 5-HT(3) receptors and altered the kinetics of 5-HT activated currents so that they resembled currents mediated by 5-HT(3AB) receptors. The 5-HT(3B) subunit also abolished the 5-HT induced [Ca(2+)](i) increase seen in untransfected NB41A3 cells. These data are consistent with the hypothesis that NB41A3 cells predominantly express homomeric 5-HT(3A) receptors that become heteromeric 5-HT(3AB) receptors upon introduction of the recombinant 5-HT(3B) subunit.

Alternative transcripts of the GABA(A) receptor epsilon subunit in human and rat.

In both human and rat tissues, complex patterns of transcripts are derived from the genes that encode the gamma-aminobutyric acid (GABA)(A) receptor epsilon subunit. An epsilon subunit transcript (approximately 3.6 kb) is expressed at relatively high levels in regions of the human brain and heart, but is not detected in most other major tissues. The encoded human epsilon subunit (epsilon (h)) confers distinctive properties to receptors into which it assembles. A distinct transcript of the gene (6.2 kb) is expressed abundantly in a variety of human tissues. This alternative transcript (ET2) appears to originate from within the epsilon subunit gene. It is possible that this transcript encodes a truncated subunit (epsilon (hS)), containing all of the transmembrane and intracellular domains. However, a combination of biochemical and electrophysiological analyses does not support this hypothesis. A distinct transcript of the epsilon subunit gene, encoding a large extracellular pro/glx domain, is expressed abundantly in rat and mouse brain. Functional analyses also failed to provide evidence for incorporation of this subunit (epsilon (rL)) into recombinant receptors. However, a shorter rat epsilon subunit (epsilon (r)), which lacks the pro/glx domain, conferred epsilon (h)-like properties to recombinant receptors, providing evidence for a functional rat epsilon subunit. In common with its human orthologue, incorporation of the epsilon (r) subunit into recombinant GABA(A) receptors confers several distinctive properties, including a reduced modulation by the anesthetic propofol and the appearance of spontaneous current.

Evidence for the formation of functionally distinct alphabetagammaepsilon GABA(A) receptors.

1. We transiently introduced the human GABA(A) receptor epsilon subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing alpha1beta3gamma2 receptors (WSS-1 cells) to establish whether the subunit competes with the gamma2 subunit for assembly into receptors. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the epsilon subunit cDNA. 2. The epsilon subunit did not change the potency of GABA: the GABA EC(50) was 34 +/- 6 microM in control WSS-1 cells and 37 +/- 6 microM in cells expressing the epsilon subunit. The introduction of the epsilon subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 +/- 0.2 nA in control cells to 0.9 +/- 0.2 nA in cells expressing the epsilon subunit (P < 0.05). 3. The epsilon subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from epsilon subunit-containing WSS-1 cells exhibited spontaneously opening GABA(A) channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the epsilon subunit did not alter the GABA-evoked single-channel cord conductance. 4. The anaesthetic 2,6-diisopropylphenol (propofol, 3 microM) and the benzodiazepine flunitrazepam (1 microM) potentiated GABA-evoked currents recorded from control cells labelled with GFP. The epsilon subunit reduced potentiation by both agents 48-96 h after transfection. 5. The introduction of the epsilon subunit had no effect on the ability of propofol (3-30 microM) relative to GABA (1 mM) to activate GABA(A) receptors in WSS-1 cells. High concentrations of propofol (> or = 100 microM) produced a more marked desensitization of GABA(A) receptor activity in WSS-1 cells transfected with cDNA for the epsilon subunit than in control cells. 6. There was no difference in the potency of Zn(2+) as an inhibitor of currents recorded from control cells (IC(50) = 165 +/- 34 microM) or cells expressing the epsilon subunit (IC(50) = 179 +/- 11 microM). 7. GABA-activated currents recorded both from control cells and cells expressing the epsilon subunit reversed in sign at the Cl- equilibrium potential and exhibited outward rectification. 8. The introduction of the epsilon subunit changes the functional properties of GABA(A) receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of alpha, beta, gamma and epsilon subunits.

5-HT(3)-receptor subunits A and B are co-expressed in neurons of the dorsal root ganglion.

The type 3 serotonin (5-HT(3)) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). Many pharmacological, behavioral, and electrophysiological studies indicate heterogeneous properties for this receptor. Although the basis for this heterogeneity is unknown, one possible explanation for these findings resides in the subunit composition of the receptor. Two 5-HT(3)-receptor subunits have been cloned: the 5-HT(3)-receptor subunit A (5-HT(3A)) and the 5-HT(3)-receptor subunit B (5-HT(3B)). Recombinant co-expression of 5-HT(3A) and 5-HT(3B) subunits produces a functional heteromeric 5-HT(3A/3B) receptor with pharmacological and electrophysiological properties different from those displayed by the 5-HT(3A) homomeric receptor. In the present report, we used in situ hybridization histochemistry to demonstrate that the 5-HT(3B) subunit is expressed in rat dorsal root ganglion (DRG) neurons. We determined with cellular resolution that 5-HT(3B) subunit mRNA was expressed in 43.2 +/- 2.8% of the total population of DRG neurons. By comparison, the 5-HT(3A) subunit was more widely expressed, with 70.0 +/- 2.8% of the total population of DRG neurons expressing this subunit. Further analyses showed that most of the neurons containing mRNA for the 5-HT(3B) subunit (91.5 +/- 3.4%) also expressed the 5-HT(3A) subunit. In contrast, nearly half the population of neurons expressing 5-HT(3A) subunit lacked (52.8 +/- 5.9%) transcripts for the 5-HT(3B) subunit. These results provide the first evidence indicating that the 5-HT(3B) subunit of the 5-HT(3) receptor is expressed in DRG and suggest that sensory neurons have the capacity to synthesize at least two structurally different 5-HT(3) receptors: a heteromeric 5-HT(3A/3B) receptor and a homomeric 5-HT(3A) receptor. Consequently, 5-HT(3) receptors with different properties might be present in peripheral and central axons of the DRG. These findings open the possibility that distinct types of 5-HT(3) receptors may be involved in perception and/or processing of sensory information. J. Comp. Neurol. 438:163-172, 2001. Published 2001 Wiley-Liss, Inc.

Evidence for expression of heteromeric serotonin 5-HT(3) receptors in rodents.

The gene and cDNAs that encode a novel subunit of rodent serotonin 5-HT(3) receptors were isolated from mouse and rat tissues. Each of the new rodent subunits shares 40% amino acid identity with the rat 5-HT(3A) subunit and 73% identity with the human 5-HT(3B) subunit. Despite a relatively low level of structural conservation, sequence analysis and functional studies suggest that the new rodent subunits are orthologues of the human 5-HT(3B) subunit. In common with homologous human receptors, rat heteromeric 5-HT(3) receptors displayed a substantially larger single-channel conductance than homomeric 5-HT(3A) receptors. In addition, the rat heteromeric receptors were less sensitive to antagonism by tubocurarine. However, in contrast to human heteromeric receptors, those of the rat displayed pronounced inward rectification of both the whole-cell and single-channel current amplitudes. Transcripts of the mouse 5-HT(3A) and 5-HT(3B) subunits are coexpressed in several cell lines that possess endogenous 5-HT(3) receptors. In addition, treatment of rat PC12 cells with nerve growth factor induced expression of both subunit mRNAs, with a similar time course for accumulation of each transcript. The combination of functional data and expression patterns is consistent with the existence of heteromeric 5-HT(3) receptors in rodent neurons.

GABA(A) receptor epsilon and theta subunits display unusual structural variation between species and are enriched in the rat locus ceruleus.

Previously, GABA(A) receptor epsilon and theta subunits have been identified only in human. Here, we describe properties of the epsilon and theta subunit genes from mouse and rat that reveal an unusually high level of divergence from their human homologs. In addition to a low level of amino acid sequence conservation ( approximately 70%), the rodent epsilon subunit cDNAs encode a unique Pro/Glx motif of approximately 400 residues within the N-terminal extracellular domain of the subunits. Transcripts of the rat epsilon subunit were detected in brain and heart, whereas the mouse theta subunit mRNA was detectable in brain, lung, and spleen by Northern blot analysis. In situ hybridization revealed a particularly strong signal for both subunit mRNAs in rat locus ceruleus in which expression was detectable from the first postnatal day. Lower levels of coexpression were also detected in other brainstem nuclei and in the hypothalamus. However, the expression pattern of theta subunit mRNA was more widespread than that of epsilon subunit, being found also in the cerebral cortex of rat pups. In contrast to primate brain, neither subunit was expressed in the hippocampus or substantia nigra. The results indicate that GABA(A) receptor epsilon and theta subunits are evolving at a much faster rate than other known GABA(A) receptor subunits and that their expression patterns and functional properties may differ significantly between species.

The 5-HT3B subunit is a major determinant of serotonin-receptor function.

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 complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

The complete genome sequence of the gastric pathogen Helicobacter pylori.

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

Identification of a gene within the tandem array of red and green color pigment genes.

The tandem array of color pigment genes on chromosome Xq28 contains nested exons of a distinct gene. This gene (termed TEX28) is composed of five exons that span almost the entire distance between the protein-coding regions of the color pigment genes and a transketolase-related gene. Although most of the TEX28 gene is repeated within the color pigment gene array, the exclusion of exon 1 from the array is predicted to restrict transcription to a single copy of the gene. The TEX28 gene encodes a polypeptide of 410 amino acid residues. This polypeptide does not display significant homology with any known proteins in public databases. Transcripts of the gene (1.8 kb) were detected in testes, but not in any other tissue examined. Color vision disorders that result from the deletion of color pigment genes should be reappraised for associated phenotypes that may derive from disruption of the TEX28 gene.

A novel class of GABAA receptor subunit in tissues of the reproductive system.

A novel subunit of the gamma-aminobutyrate, type A (GABAA) receptor family has been identified in human and rat tissues. The subunit displays 30-40% amino acid identity with known family members and represents a distinct subunit class (termed pi). Transcripts of the pi subunit were detected in several human tissues and were particularly abundant in the uterus. The pi subunit protein can assemble with known GABAA receptor subunits and confer unique ligand binding properties to the recombinant receptors in which it combines. Most notably, the presence of the pi subunit alters the sensitivity of recombinant receptors to the endogenous steroid, pregnanolone. Identification of the pi subunit indicates a new target for pharmacological manipulation of GABAA receptors that are located outside of the central nervous system.

Human pyridoxal kinase. cDNA cloning, expression, and modulation by ligands of the benzodiazepine receptor.

Peptide fragments of a porcine benzodiazepine-binding protein were used to isolate the cDNA of a related human protein. The cDNA encodes a polypeptide of 312 amino acid residues that is homologous to a bacterial pyridoxal kinase. Transient expression of the cDNA in human embryonic kidney cells confirmed that it encodes human pyridoxal kinase. The recombinant enzyme displayed a Km value of 3.3 microM for pyridoxal and was inhibited competitively by 4-deoxypyridoxine (Ki = 2.8 microM). Benzodiazepine receptor ligands that bound to the purified porcine protein also exerted a potent inhibitory effect on human pyridoxal kinase activity. Transcripts of the pyridoxal kinase gene were detectable in all human tissues examined, and were particularly abundant in the testes. The gene is localized on chromosome 21q22.3 and represents a candidate gene for at least one genetic disorder that has been mapped to this region (autoimmune polyglandular disease type 1).

Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells.

1. Radioligand binding and patch-clamp techniques were used to study the actions of gamma-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5 alpha-pregnan-3 alpha-ol-20-one on rat alpha 1 and beta 3 GABAA receptor subunits, expressed either alone or in combination. 2. Membranes from HEK293 cells after transfection with alpha 1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (< 0.03 pmol mg-1 protein). GABA (100 microM) applied to whole-cells transfected with alpha 1 cDNA and clamped at -60 mV, also failed to activate discernible currents. 3. The membranes of cells expressing beta 3 cDNAs bound [35S]-TBPS (approximately 1 pmol mg-1 protein). However, the binding was not influenced by GABA (10 nM-100 microM). Neither GABA (100 microM) nor picrotoxin (10 microM) affected currents recorded from cells expressing beta 3 cDNA, suggesting that beta 3 subunits do not form functional GABAA receptors or spontaneously active ion channels. 4. GABA (10 nM-100 microM) modulated [35S]-TBPS binding to the membranes of cells transfected with both alpha 1 and beta 3 cDNAs. GABA (0.1 microM-1 mM) also dose-dependently activated inward currents with an EC50 of 9 microM recorded from cells transfected with alpha 1 and beta 3 cDNAs, clamped at -60 mV. 5. Propofol (10 nM-100 microM), pentobarbitone (10 nM-100 microM) and 5 alpha-pregnan-3 alpha-ol-20-one (1 nM-30 microM) modulated [35S]-TBPS binding to the membranes of cells expressing either alpha 1 beta 3 or beta 3 receptors. Propofol (100 microM), pentobarbitone (1 mM) and 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) also activated currents recorded from cells expressing alpha 1 beta 3 receptors. 6. Propofol (1 microM-1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing beta 3 homomers. In contrast, application of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) failed to activate detectable currents. 7. Propofol (100 microM)-activated currents recorded from cells expressing either alpha 1 beta 3 or beta 3 receptors reversed at the Cl- equilibrium potential and were inhibited to 34 +/- 13% and 39 +/- 10% of control, respectively, by picrotoxin (10 microM). 5 alpha-Pregnan-3 alpha-ol-20-one (100 nM) enhanced propofol (100 microM)-evoked currents mediated by alpha 1 beta 3 receptors to 1101 +/- 299% of control. In contrast, even at high concentration 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) caused only a modest facilitation (to 128 +/- 12% of control) of propofol (100 microM)-evoked currents mediated by beta 3 homomers. 8. Propofol (3-100 microM) activated alpha 1 beta 3 and beta 3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 microM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a "surge' current on cessation of propofol (300 microM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) to cells expressing alpha 1 beta 3 receptors. 9. These observations demonstrate that rat beta 3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABAA receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.