Inhibitory neurotransmission mediated by GABA(A) receptors can be modulated by the endogenous neurosteroids, allopregnanolone and tetrahydro-deoxycorticosterone. Neurosteroids are synthesized de novo in the brain during stress, pregnancyand after ethanol consumption, and disrupted steroid regulation of GABAergic transmission is strongly implicated in several debilitating conditions such as panic disorder, major depression, schizophrenia, alcohol dependence and catamenial epilepsy. Determining how neurosteroids interact with the GABA(A) receptor is a prerequisite for understanding their physiological and pathophysiological roles in the brain. Here we identify two discrete binding sites in the receptor's transmembrane domains that mediate the potentiating and direct activation effects of neurosteroids. They potentiate GABA responses from a cavity formed by the alpha-subunit transmembrane domains, whereas direct receptor activation is initiated by interfacial residues between alpha and beta subunits and is enhanced by steroid binding to the potentiation site. Thus, significant receptor activation by neurosteroids relies on occupancy of both the activation and potentiation sites. These sites are highly conserved throughout the GABA(A )receptor family, and their identification provides a unique opportunity for the development of new therapeutic, neurosteroid-based ligands and transgenic disease models of neurosteroid dysfunction.

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GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

Slow and persistent synaptic inhibition is mediated by metabotropic GABA B receptors (GABA B Rs). GABA B Rs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABA B Rs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABA B Rs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABA B Rs. These sorting events are paralleled by a reduction in GABA B R-dependent activation of inwardly rectifying K + channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABA B R2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABA B Rs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABA B Rs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.he availability of neurotransmitter receptors, a major determinant of synaptic efficacy, is regulated by coordinated mechanisms of intracellular trafficking that deliver newly synthesized receptors to the plasma membrane and remove them for storage, recycling, or degradation (1). The molecular mechanisms controlling the availability of GABA B receptors (GABA B Rs), which are central players in the modulation of excitatory and inhibitory synaptic activity, are unclear.GABA B Rs mediate slow and prolonged inhibitory synaptic signals (2, 3). Consistent with these roles, modifications in the function of GABA B Rs are implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, cognition, and addictive mechanisms to drugs of abuse (3-7). GABA B Rs are members of the G protein-coupled receptor (GPCR) superfamily and are obligatory heteromers composed of two related subunits, namely GABA B R1 and GABA B R2 (3, 8). GABA B R1 binds agonist with high affinity, whereas GABA B R2 mediates coupling to Gαi (9, 10). GABA B Rs are located in GABA-ergic and glutamatergic pre-and postsynaptic terminals, but their distribution does not coincide with the active zone, postsynaptic density, or inhibitory postsynaptic specializations. Rather, they are perisynaptic receptors activated by GABA spillover (3, 11). Stimulation of GABA B Rs decreases the levels of cAMP, inhibits neurotr...

The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world. However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive. Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity. Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES). Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects. For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects. Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases. Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity. We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens.

GABA B receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre-and postsynaptic sites, GABA B receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA B receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, ␣-bungarotoxin. By using the ␣-bungarotoxin binding site-tagged GABA B R1a subunit (R1a BBS ), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, ␣-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA B receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors. ␥-Aminobutyric acid (GABA)2 is the major inhibitory neurotransmitter in the central nervous system (CNS) activating ionotropic GABA A/C , as well as the metabotropic GABA B receptor. GABA B receptors are expressed in all major brain structures (1-3) and are important for synaptic plasticity as well as having therapeutic implications for epilepsy, pain, spasticity, drug addiction, schizophrenia, depression, and anxiety (4).The trafficking and cell surface mobility of ligand-gated GABA A receptors has been studied using reporter tags with electrophysiological (5) or imaging approaches (6, 7). However, the mobility and trafficking of extrasynaptic GABA B receptors has provided diverse results (8 -11). The GABA B receptor is a heterodimeric G-protein-coupled receptor (GPCR), requiring R1 and R2 subunits to co-assemble before trafficking to the cell surface to form functional receptors. The R1 subunit possesses an ER retention motif that is masked by binding to the R2 subunit (12-14). Although, generally, GPCRs are readily internalized from the cell surface following agonist activation and receptor phosphorylation (15-17); the GABA B receptor was thought to behave differently, being relatively stable in the cell membrane (8, 9). However, other reports indicate that agonist activation of GABA B receptors may promote internalization and/or rapid recycling (10,11,18). To address the topic of GABA B receptor trafficking, prior studies have used various techniques to monitor receptor movement, including: receptor biotinylation (8, 9); antibody labeling of extracellular GABA B receptor epitopes on live cells (9); as well as fluorescence recovery after photobleaching (FRAP) (10). These methods have therefore relied on the use of relatively large reporter molecules, such as antibodies. Although such studies have...

GABA type A (GABA A ) receptors are functionally regulated by external protons in a manner dependent on the receptor subunit composition. Although H ϩ can regulate the open probability of single GABA ion channels, exactly what residues and receptor subunits are responsible for proton-induced modulation remain unknown. This study resolves this issue by using recombinant ␣1␤i subunit GABA A receptors expressed in human embryonic kidney cells. The potentiating effect of low external pH on GABA responses exhibited p Ka in accord with the involvement of histidine and/or cysteine residues. The exposure of GABA A receptors to the histidine-modifying reagent DEPC ablated regulation by H ϩ , implicating the involvement of histidine residues rather than cysteines in proton regulation. Site-specific substitution of all conserved external histidines to alanine on the ␤ subunits revealed that H267 alone, in the TM2 domain, is important for H ϩ regulation. These results are interpreted as a direct protonation of H267 on ␣1␤i receptors rather than an involvement in signal transduction. The opposing functional effects induced by Zn 2ϩ and H ϩ at this single histidine residue most likely reflect differences in charge delocalization on the imidazole rings in the mouth of the GABA A receptor ion channel. Additional substitutions of H267 in ␤ subunits with other residues possessing charged side chains (glutamate and lysine) reveal that this area of the ion channel can profoundly influence the functional properties of GABA A receptors.

GABA B receptors mediate slow inhibitory neurotransmission in the brain and feature during excitatory synaptic plasticity, as well as various neurological conditions. These receptors are obligate heterodimers composed of GABA B R1 and R2 subunits. The two predominant R1 isoforms differ by the presence of two complement control protein modules or Sushi domains (SDs) in the N terminus of R1a. By using live imaging, with an α-bungarotoxin-binding site (BBS) and fluorophore-linked bungarotoxin, we studied how R2 stabilizes R1b subunits at the cell surface. Heterodimerization with R2 reduced the rate of internalization of R1b, compared with R1b homomers. However, R1aR2 heteromers exhibited increased cell surface stability compared with R1bR2 receptors in hippocampal neurons, suggesting that for receptors containing the R1a subunit, the SDs play an additional role in the surface stability of GABA B receptors. Both SDs were necessary to increase the stability of R1aR2 because single deletions caused the receptors to be internalized at the same rate and extent as R1bR2 receptors. Consistent with these findings, a chimera formed from the metabotropic glutamate receptor (mGluR)2 and the SDs from R1a increased the surface stability of mGluR2. These results suggest a role for SDs in stabilizing cell surface receptors that could impart different pre-and postsynaptic trafficking itineraries on GABA B receptors, thereby contributing to their physiological and pathological roles. underlying slow GABA-mediated inhibitory neurotransmission (1) that regulates neuronal excitability (2-4). These receptors are increasingly considered as potential therapeutic targets for a range of diseases, including epilepsy, schizophrenia, anxiety, depression, and substance abuse (1, 5). Functional GABA B receptors are heteromers formed from GABA B R1 subunits containing the agonist binding domain (6) and R2 subunits that link to G protein signaling (7,8).To date, just one isoform of the R2 subunit has been reported (9, 10), whereas several isoforms of the R1 subunit exist [R1a, R1b, R1c, R1e, R1j (1)]. Among these, R1a and R1b predominate in the central nervous system (CNS), and their expression arises by the use of different promoters (11) of the GABBR1 gene. R1a subunits contain 143 aa forming two Sushi domains (SDs) in the N terminus, which are also known as complement control proteins or short consensus repeats (12). These SDs are absent in R1b being replaced by 18 unique amino acids.Heteromers formed from R1aR2 and R1bR2 are thought to play distinct roles in neurotransmission (13-18), with R1aR2 contributing to presynaptic heteroreceptors to inhibit neurotransmitter release, although both R1aR2 and R1bR2 populate postsynaptic membranes to dampen excitability. These heteromeric subtypes exhibit different subcellular compartmentalization with the SDs acting as an axonal-targeting sequence to deliver R1aR2 more efficiently to axons compared with R1bR2 (19). This would, in part, explain the differential pre-and postsynaptic signaling properties o...