A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat γ2 or γ3, in combination with α1 and β2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a γ3 subunit display a 178-fold reduced affinity to zolpidem as compared with γ2-containing receptors. Eight chimeras between γ2 and γ3 were constructed followed by nine different point mutations in γ2, each to the homologous amino acid residue found in γ3. Chimeric or mutant γ subunits were coexpressed with α1 and β2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ2 (γ2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Dopamine receptor subtypes modulate olfactory bulb γ-aminobutyric acid type A receptors

The γ-aminobutyric acid type A (GABAA) receptor is the predominant Cl− channel protein mediating inhibition in the olfactory bulb and elsewhere in the mammalian brain. The olfactory bulb is rich in neurons containing both GABA and dopamine. Dopamine D1 and D2 receptors are also highly expressed in this brain region with a distinct and complementary distribution pattern. This distribution suggests that dopamine may control the GABAergic inhibitory processing of odor signals, possibly via different signal-transduction mechanisms. We have observed that GABAA receptors in the rat olfactory bulb are differentially modulated by dopamine in a cell-specific manner. Dopamine reduced the currents through GABA-gated Cl- channels in the interneurons, presumably granule cells. This action was mediated via D1 receptors and involved phosphorylation of GABAA receptors by protein kinase A. Enhancement of GABA responses via activation of D2 dopamine receptors and phosphorylation of GABAA receptors by protein kinase C was observed in mitral/tufted cells. Decreasing or increasing the binding affinity for GABA appears to underlie the modulatory effects of dopamine via distinct receptor subtypes. This dual action of dopamine on inhibitory GABAA receptor function in the rat olfactory bulb could be instrumental in odor detection and discrimination, olfactory learning, and ultimately odotopic memory formation.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

A study was made of glycine (Gly) and γ-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 ± 35, 346 ± 28, and 389 ± 22 nA, whereas the GABA-currents (1 mM GABA) were all ≤40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC50 for Gly was 611 ± 77 μM for encephalon, 661 ± 28 μM for midbrain, and 506 ± 18 μM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all ≈2. Strychnine inhibited all of the Gly-currents, with an IC50 of 56 ± 3 nM for encephalon, 97 ± 4 nM for midbrain, and 72 ± 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca2+ concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription–PCR studies showed differences in the Gly receptor (GlyR) α-subunits expressed, whereas the β-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca2+ could play important functions during brain development.

Brain lipids that induce sleep are novel modulators of 5-hydroxytrypamine receptors.

Amide derivatives of fatty acids were recently isolated from cerebrospinal fluid of sleep-deprived animals and found to induce sleep in rats. To determine which brain receptors might be sensitive to these novel neuromodulators, we tested them on a range of receptors expressed in Xenopus oocytes. cis-9,10-Octadecenamide (ODA) markedly potentiated the action of 5-hydroxytryptamine (5-HT) on 5-HT2A and 5-HT2C receptors, but this action was not shared by related compounds such as oleic acid and trans-9,10-octacenamide. ODA was active at concentrations as low as 1 nM. The saturated analog, octadecanamide, inhibited rather than potentiated 5-HT2C responses. ODA had either no effect or only weak effects on other receptors, including muscarinic cholinergic, metabotropic glutamate, GABA(A), N-methyl-D-asparate, or alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid receptors. Modulation of 5-HT2 receptors by ODA and related lipids may represent a novel mechanism for regulation of receptors that activate G proteins and thereby play a role in alertness, sleep, and mood as well as disturbances of these states.

Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.

A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition.

A class of bicuculline-insensitive gamma-aminobutyric acid (GABA) receptors, GABAC, has been identified in retina. Several lines of evidence indicate that GABAC receptors are formed partially or wholly of GABA rho subunits. These receptors generate a Cl- current in response to GABA but differ from GABAA receptors in a number of ways. Picrotoxin, widely accepted as a noncompetitive antagonist of GABAA receptors, displays competitive and noncompetitive antagonism of GABAC receptors in perch and bovine retina and GABA rho 1 receptors expressed in Xenopus oocytes. The aim of this study was to identify the molecular basis of the two components of picrotoxin inhibition of GABA rho 1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between rho 1 and rho 2 receptors was localized to a single amino acid in the putative second transmembrane domain. Substitution of this amino acid with residues found in the analogous position in highly picrotoxin-sensitive glycine alpha and GABAA subunits increased the sensitivity of rho 1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the rho 1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA rho 1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an extemal I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Glycine receptors: A new therapeutic target in pain pathways

Although glycine receptor Cl- channels (GlyRs) have long been known to mediate inhibitory neurotransmission onto spinal nociceptive neurons, their therapeutic potential for peripheral analgesia has received little attention. However, it has been shown that alpha 3-subunit-containing GlyRs are concentrated into regions of the spinal cord dorsal horn where nociceptive afferents terminate. Furthermore, inflammatory mediators specifically inhibit alpha 3-containing GlyRs, and deletion of the murine alpha 3 gene confers insensitivity to chronic inflammatory pain. This strongly implicates GlyRs in the inflammation-mediated disinhibition of centrally projecting nociceptive neurons. Future therapies aimed at specifically increasing current flux through alpha 3-containing GlyRs may prove effective in providing analgesia.

Functional CB2 type cannabinoid receptors at CNS synapses

To date, it has been thought that cannabinoid receptors in CNS are primarily of the CB1R subtype, with CB2R expressed only in glia and peripheral tissues. However, evidence for the expression of CB2 type cannabinoid receptors at neuronal sites in the CNS is building through anatomical localization of receptors and mRNA in neurons and behavioural studies of central effects of CB2R agonists. In the medial entorhinal area of the rat, we found that blockade of CB1R did not occlude suppression of GABAergic inhibition by the non-specific endogenous cannabinoid 2-AG, suggesting that CB1R could not account fully for the effects of 2-AG. Suppression could be mimicked using the CB2R agonist JWH-133 and reversed by the CB2R inverse agonist AM-630, indicating the presence of functional CB2R. When we reversed the order of drug application AM-630 blocked the effects of the CB2R agonist JWH-133, but not the CB1R inverse agonist LY320135. JTE-907, a CB2R inverse agonist structurally unrelated to AM-630 elicited increased GABAergic neurotransmission at picomolar concentrations. Analysis of mIPSCs revealed that CB2R effects were restricted to action potential dependent, but not action potential independent GABA release. These data provide pharmacological evidence for functional CB2R at CNS synapses.

Background synaptic activity in rat entorhinal cortical neurones:differential control of transmitter release by presynaptic receptors

The entorhinal cortex (EC) is a key brain area controlling both hippocampal input and output via neurones in layer II and layer V, respectively. It is also a pivotal area in the generation and propagation of epilepsies involving the temporal lobe. We have previously shown that within the network of the EC, neurones in layer V are subject to powerful synaptic excitation but weak inhibition, whereas the reverse is true in layer II. The deep layers are also highly susceptible to acutely provoked epileptogenesis. Considerable evidence now points to a role of spontaneous background synaptic activity in control of neuronal, and hence network, excitability. In the present article we describe results of studies where we have compared background release of the excitatory transmitter, glutamate, and the inhibitory transmitter, GABA, in the two layers, the role of this background release in the balance of excitability, and its control by presynaptic auto- and heteroreceptors on presynaptic terminals. © The Physiological Society 2004.

Analysis Of The Contribution Of Immunologically-detectable Her2, Steroid Receptors And Of The Triple-negative" Tumor Status To Disease-free And Overall Survival Of Women With Epithelial Ovarian Cancer."

We assessed associations between steroid receptors including: estrogen-alpha, estrogen-beta, androgen receptor, progesterone receptor, the HER2 status and triple-negative epithelial ovarian cancer (ERα-/PR-/HER2-; TNEOC) status and survival in women with epithelial ovarian cancer. The study included 152 women with primary epithelial ovarian cancer. The status of steroid receptor and HER2 was determined by immunohistochemistry. Disease-free and overall survival were calculated and compared with steroid receptor and HER2 status as well as clinicopathological features using the Cox Proportional Hazards model. A mean follow-up period of 43.6 months (interquartile range=41.4 months) was achieved where 44% of patients had serous tumor, followed by mucinous (23%), endometrioid (9%), mixed (9%), undifferentiated (8.5%) and clear cell tumors (5.3%). ER-alpha staining was associated with grade II-III tumors. Progesterone receptor staining was positively associated with a Body Mass Index≥25. Androgen receptor positivity was higher in serous tumors. In stand-alone analysis of receptor contribution to survival, estrogen-alpha positivity was associated with greater disease-free survival. However, there was no significant association between steroid receptor expression, HER2 status, or TNEOC status, and overall survival. Although estrogen-alpha, androgen receptor, progesterone receptor and the HER2 status were associated with key clinical features of the women and pathological characteristics of the tumors, these associations were not implicated in survival. Interestingly, women with TNEOC seem to fare the same way as their counterparts with non-TNEOC.

Inflammatory Sensitization Of Nociceptors Depends On Activation Of Nmda Receptors In Drg Satellite Cells.

The present study evaluated the role of N-methyl-D-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E2 (PGE2), which was significantly attenuated by injection of the NMDAR antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE2-induced hyperalgesia in the paw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE2-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE2-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.