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1.
BackgroundGABA A receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs [1]. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear [2]. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die [3]– [6]. As many anaesthetics act via GABA A receptors [7], the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. Principal FindingsWe demonstrate, using RT-PCR, that monocytes express GABA A receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA A receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA A receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SignificanceOur results show that functional GABA A receptors are present on monocytes with properties similar to CNS GABA A receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA A receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA A receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem. 相似文献
2.
PRIP, phospholipase C related, but catalytically inactive protein was first identified as a novel inositol 1,4,5-trisphosphate binding protein. It has a number of binding partners including protein phosphatase (PP1 and 2A), GABA A receptor associated protein, and the β subunits of GABA A receptors, in addition to inositol 1,4,5-trisphosphate. The identification of these molecules led us to examine the possible involvement of PRIP in the phospho-regulation of the β subunits of GABA A receptors using hippocampal neurons prepared from PRIP-1 and 2 double knock-out (DKO) mice. Experiments were performed with special reference to the dephosphorylation processes of the β subunits. The phosphorylation of β3 subunits by the activation of protein kinase A in cortical neurons of the control mice continued for up to 5 min, even after washing out of the stimulus, followed by a gradual dephosphorylation. That of DKO mice gradually increased in spite of the lower phosphorylation levels induced by the stimulation. There was little difference in the amount of cellular cyclic AMP and protein kinase A activity between the control and mutant mice, indicating that phosphatases such as PP1 and PP2A are primarily involved in the difference. The time course of PP1 activity changes in the vicinity of the receptors in control mice corresponded to the phosphorylation of PRIP, while that of the mutant mice decreased with the period of the incubation. This is a good agreement with the suggestion that PRIP binds to and inactivates PP1, which is regulated by the phosphorylation of PRIP at threonine 94. These results suggest that PRIP plays an important role in controlling the dynamics of GABA A receptor phosphorylation by through PP1 binding and, therefore, the efficacy of synaptic inhibition mediated by these receptors. 相似文献
3.
Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABA A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA A response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABA A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA A receptors. 相似文献
4.
Propofol, an intravenous anesthetic, is a positive modulator of the GABA A receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3 H-diazirin-3-yl)phenol, AziP m-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABA A receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABA A receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABA A receptor subunit protection. This investigation demonstrates striking interfacial GABA A receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. 相似文献
5.
GABA A receptor subunit composition is a critical determinant of receptor localization and physiology, with synaptic receptors generating phasic inhibition and extrasynaptic receptors producing tonic inhibition. Extrasynaptically localized α5 GABA A receptors are largely responsible for tonic inhibition in hippocampal neurons. However, we show here that inhibitory synapses also contain a constant level of α5 GABA A receptors throughout neuronal development, as measured by its colocalization with gephyrin, the inhibitory postsynaptic scaffolding protein. Immunoprecipitation of the α5 subunit from both cultured neurons and adult rat brain coimmunoprecipitated gephyrin, confirming this interaction in vivo. Furthermore, the α5 subunit can interact with gephyrin independent of other synaptically localized alpha subunits, as shown by immunoprecipitation experiments in HEK cells. By replacing the α5 predicted gephyrin binding domain (Residues 370–385) with either the high affinity gephyrin binding domain of the α2 subunit or homologous residues from the extrasynaptic α4 subunit that does not interact with gephyrin, α5 GABA A receptor localization shifted into or out of the synapse, respectively. These shifts in the ratio of synaptic/extrasynaptic α5 localization disrupted dendritic outgrowth and spine maturation. In contrast to the predominant view of α5 GABA A receptors being extrasynaptic and modulating tonic inhibition, we identify an intimate association of the α5 subunit with gephyrin, resulting in constant synaptic levels of α5 GABA AR throughout circuit formation that regulates neuronal development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1241–1251, 2015 相似文献
6.
CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABA A receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABA A receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABA A receptor clusters in hippocampal neurons. 相似文献
7.
The GABA A-receptor-associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and/or recycling of GABA A receptors. GABARAP is localized on intracellular membranes such as the trans-Golgi network, binds to the γ 2 subunit of GABA A receptors and interacts with microtubules and the N-ethylmaleimide-sensitive factor. We report the X-ray crystal structure of mammalian GABARAP at 2.0 Å resolution. GABARAP consists of an N-terminal basic helical region, which has been implicated in tubulin binding, and a core structure with a conserved ubiquitin-like fold. Consistent with the high extent of sequence conservation among GABARAP homologues from plants to mammals, one face of the core structure is absolutely conserved while the opposite face shows considerable divergence. These features are in agreement with the conserved surface mediating protein–protein interactions shared by all members of the family, whereas the non-conserved surface region may play specific roles, such as docking to particular membrane receptors. 相似文献
8.
Gephyrin and collybistin are key components of GABA A receptor (GABA AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA AR α3 interacts with gephyrin, GABA AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA AR α2 and collybistin or GABA AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA ARs, but not GlyRs or other GABA AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA ARs containing the α2 subunit. 相似文献
9.
Synaptic scaling represents a process whereby the distribution of a cell''s synaptic strengths are altered by a multiplicative scaling factor. Scaling is thought to be a compensatory response that homeostatically controls spiking activity levels in the cell or network. Previously, we observed GABAergic synaptic scaling in embryonic spinal motoneurons following in vivo blockade of either spiking activity or GABA A receptors (GABA ARs). We had determined that activity blockade triggered upward GABAergic scaling through chloride accumulation, thus increasing the driving force for these currents. To determine whether chloride accumulation also underlies GABAergic scaling following GABA AR blockade we have developed a new technique. We expressed a genetically encoded chloride-indicator, Clomeleon, in the embryonic chick spinal cord, which provides a non-invasive fast measure of intracellular chloride. Using this technique we now show that chloride accumulation underlies GABAergic scaling following blockade of either spiking activity or the GABA AR. The finding that GABA AR blockade and activity blockade trigger scaling via a common mechanism supports our hypothesis that activity blockade reduces GABA AR activation, which triggers synaptic scaling. In addition, Clomeleon imaging demonstrated the time course and widespread nature of GABAergic scaling through chloride accumulation, as it was also observed in spinal interneurons. This suggests that homeostatic scaling via chloride accumulation is a common feature in many neuronal classes within the embryonic spinal cord and opens the possibility that this process may occur throughout the nervous system at early stages of development. 相似文献
10.
Low levels of docosahexaenoic acid (DHA) have been linked to a number of mental illnesses such as memory loss, depression and schizophrenia. While supplementation of DHA is beneficial in improving memory and cognition, the influence of dietary fats on the neurotransmitters and receptors involved in cognitive function is still not known. The aim of this study was to investigate serotonin receptor (5-HT 1A and 5-HT 2A), cannabinoid receptor (CB1) and gamma-aminobutyric acid type A (GABA A) receptor binding densities in the brain of male rats fed a high-saturated-fat (HF) diet, as well as the effect of DHA supplementation on HF diet. Alterations of these receptors in the post-mortem rat brain were detected by [ 3H]-WAY-100635, [ 3H]-ketanserin, [ 3H]-CP-55,940 and [ 3H]-muscimol binding autoradiography, respectively. In the hippocampus, the 5-HT 1A, CB1 and GABA A receptor binding densities significantly increased in response to an HF diet, while in the hypothalamus, 5-HT 1A and CB1 binding densities significantly increased in HF-fed rats. Importantly, DHA supplementation prevented the HF-induced increase of receptors binding density in the hippocampus and hypothalamus. Furthermore, DHA supplementation attenuated 5-HT 2A receptor binding density in the caudate putamen, anterior cingulate cortex and medial mammillary nucleus, which was also increased in HF group. This study showed that an HF diet increased 5-HT 1A, 5-HT 2A, CB1 and GABA A receptor binding densities in the brain regions involved in cognitive function and that dietary DHA can attenuate such alterations. These findings provide insight into the mechanism by which DHA supplementation ameliorates reduced cognitive function associated with an HF diet. 相似文献
12.
Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine.In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5, 10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid ( μ, δ, k), cannabinoid (CB 1, CB 2), glutamatergic (NMDA), GABAergic (GABA A, GABA B), serotoninergic (5HT 2A) and adrenergic (α 2) receptors.The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine-induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the GABA A and moderate affinity for GABA B, NMDA and δ opioid receptors.WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of GABA A, GABA B, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies. 相似文献
13.
The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of γ-aminobutyric acid A receptor (GABA AR) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 μg/ml of tunicamycin for 24 h blocked the binding of 3H-flunitrazepam and 3H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABA AR immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by 3H-flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABA AR by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin-treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of 3H-sugars and 3H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 μg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABA AR α 1-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA AR by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation may be important for the functional expression and intracellular transport of GABA AR. J. Cell. Biochem. 70:38–48, 1998. © 1998 Wiley-Liss, Inc. 相似文献
14.
The γ-aminobutyric acid type A (GABA A) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA A receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 Å resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA A receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein. 相似文献
15.
Extracts from Glycyrrhiza are traditionally used for the treatment of insomnia and anxiety. Glabridin is one of the main flavonoid compounds from Glycyrrhiza glabra and displays a broad range of biological properties. In the present work, we investigated the effect of glabridin on GABA A receptors. For this purpose, we employed the two-electrode voltage-clamp technique on Xenopus laevis oocytes expressing recombinant GABA A receptors. Through this approach, we observed that glabridin presents a strong potentiating effect on GABA A α1β(1?3)γ2 receptors. The potentiation was slightly dependent on the β subunit and was most pronounced at the α1β2γ2 subunit combination, which forms the most abundant GABA A receptor in the CNS. Glabridin potentiated with an EC 50 of 6.3±1.7 µM and decreased the EC 50 of the receptor for GABA by approximately 12-fold. The potentiating effect of glabridin is flumazenil-insensitive and does not require the benzodiazepine binding site. Glabridin acts on the β subunit of GABA A receptors by a mechanism involving the M286 residue, which is a key amino acid at the binding site for general anesthetics, such as propofol and etomidate. Our results demonstrate that GABA A receptors are strongly potentiated by one of the main flavonoid compounds from Glycyrrhiza glabra and suggest that glabridin could contribute to the reported hypnotic effect of Glycyrrhiza extracts. 相似文献
16.
Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABAA receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABAA communication between astrocytes and neurons. The effects of two putative agonists of GABAA, β-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABAA receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response. 相似文献
17.
GABA A receptors (GABA ARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein–protein interactions that maintain the appropriate number of GABA ARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABA ARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABA ARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABA ARs at the neuronal surface, but had no effect on the total amount of GABA ARs. Inhibition of GABA AR endocytosis by muscimol increased both GABA ARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABA ARs. Those data suggest an important function of BIG1 in trafficking of GABA ARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl ? influx through the regulation of cell surface expression of GABA ARs. 相似文献
18.
Modulation of the GABA type A receptor (GABA AR) function by cholesterol and other steroids is documented at the functional level, yet its structural basis is largely unknown. Current data on structurally related modulators suggest that cholesterol binds to subunit interfaces between transmembrane domains of the GABA AR. We construct homology models of a human GABA AR based on the structure of the glutamate-gated chloride channel GluCl of Caenorhabditis elegans. The models show the possibility of previously unreported disulfide bridges linking the M1 and M3 transmembrane helices in the α and γ subunits. We discuss the biological relevance of such disulfide bridges. Using our models, we investigate cholesterol binding to intersubunit cavities of the GABA AR transmembrane domain. We find that very similar binding modes are predicted independently by three approaches: analogy with ivermectin in the GluCl crystal structure, automated docking by AutoDock, and spontaneous rebinding events in unbiased molecular dynamics simulations. Taken together, the models and atomistic simulations suggest a somewhat flexible binding mode, with several possible orientations. Finally, we explore the possibility that cholesterol promotes pore opening through a wedge mechanism. 相似文献
19.
(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABA A receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABA A receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyloxycarbonyl)-3,4-epoxypiperidine-4-carboxylate ( 1 ) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee ≥ 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [ 3H]GABA to GABA B receptor sites (IC 50 > 100 μ M), (+)-isoguvacine oxide (IC 50 = 0.20 ± 0.03 μ M) and (?)-isoguvacine oxide (IC 50 = 0.32 ± 0.05 μ M) showed comparable potencies as inhibitors of the binding of [ 3H]GABA to GABA A receptor sites. Furthermore, (+)-isoguvacine oxide (EC 50 = 6 μ M; 33% relative efficacy) and (?)-isoguvacine oxide (EC 50 = 5 μ M; 38% efficacy relative to 10 μ M muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [ 3H]diazepam to the GABA A receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABA A agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABA A recognition site(s), derived from extensive structure—activity studies on GABA A agonists. Thus, the model of the GABA A recognition site(s) comprising a narrow cleft or pocket, in which the anionic moiety of the zwitterionic GABA A agonists is assumed to be embedded during receptor activation, may have to be revised. © 1995 Wiley-Liss, Inc. 相似文献
20.
Photoaffinity labeling of γ-aminobutyric acid type A
(GABA A)-receptors (GABA AR) with an etomidate analog and
mutational analyses of direct activation of GABA AR by neurosteroids
have each led to the proposal that these structurally distinct general
anesthetics bind to sites in GABA ARs in the transmembrane domain at
the interface between the β and α subunits. We tested whether the
two ligand binding sites might overlap by examining whether neuroactive
steroids inhibited etomidate analog photolabeling. We previously identified
(Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and
Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate
photolabeling of GABA AR α1Met-236 and βMet-286 (in
αM1 and βM3). Positioning these two photolabeled amino acids in a
single type of binding site at the interface of β and α subunits
(two copies per pentamer) is consistent with a GABA AR homology
model based upon the structure of the nicotinic acetylcholine receptor and
with recent αM1 to βM3 cross-linking data. Biologically active
neurosteroids enhance rather than inhibit azietomidate photolabeling, as
assayed at the level of GABA AR subunits on analytical SDS-PAGE, and
protein microsequencing establishes that the GABA AR-modulating
neurosteroids do not inhibit photolabeling of GABA AR
α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus
modulatory steroids do not bind at the same site as etomidate, and neither of
the amino acids identified as neurosteroid activation determinants (Hosie, A.
M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature
444, 486–489) are located at the subunit interface defined by our
etomidate site model.GABA A3
receptors (GABA AR) are major mediators of brain inhibitory
neurotransmission and participate in most circuits and behavioral pathways
relevant to normal and pathological function
( 1). GABA AR are
subject to modulation by endogenous neurosteroids, as well as myriad
clinically important central nervous system drugs including general
anesthetics, benzodiazepines, and possibly ethanol
( 1,
2). The mechanism of
GABA AR modulation by these different classes of drugs is of major
interest, including identification of the receptor amino acid residues
involved in binding and action of the drugs.In the absence of high resolution crystal structures of drug-receptor
complexes, the locations of anesthetic binding sites in GABA ARs
have been predicted based upon analyses of functional properties of point
mutant receptors, which identified residues in the α and β subunit
M1–M4 transmembrane helices important for modulation by volatile
anesthetics (primarily α subunit) and by intravenous agents, including
etomidate and propofol (β subunit)
( 3– 5).
Position βM2–15, numbered relative to the N terminus of the helix,
functions as a major determinant of etomidate and propofol potency as GABA
modulators in vitro and in vivo
( 6– 8).
By contrast, this residue is not implicated for modulation by the
neurosteroids, potent endogenous modulators of GABA AR
( 9).Photoaffinity labeling, which allows the identification of residues in
proximity to drug binding sites
( 10,
11), has been used to identify
two GABA AR amino acids covalently modified by the etomidate analog
[ 3H]azietomidate
( 12): α1Met-236 within
αM1 and βMet-286 within βM3. Photolabeling of these residues
was inhibited equally by nonradioactive etomidate and enhanced proportionately
by GABA present in the assay, consistent with the presence of these two
residues in a common drug binding pocket that would be located at the
interface between the β and α subunits in the transmembrane domain
( 12). Mutational analyses
identify these positions as etomidate and propofol sensitivity determinants
( 13– 15).A recent mutagenesis study
( 16) identified two other
residues in GABA AR αM1 and βM3 as critical for direct
activation by neurosteroids, αThr-236 (rat numbering, corresponding to
α1Thr-237, bovine numbering used here and by Li et al.
( 12)) 4
and βTyr-284. These residues were also proposed to contribute to a
neurosteroid binding pocket in the transmembrane domain at the interface
between β and α subunits, based upon their location in an
alternative GABA AR structural model that positioned those amino
acids, and not α1Met-236 or βMet-286, at the subunit interface. For
GABA ARs and other members of the Cys-loop superfamily of
neurotransmitter-gated ion channels, the transmembrane domain of each subunit
is made up of a loose bundle of four α helices (M1–M4), with M2
from each subunit contributing to the lumen of the ion channel and M4
positioned peripherally in greatest contact with lipid, as seen in the
structures of the Torpedo nicotinic acetylcholine receptor (nAChR)
( 17) and in distantly related
prokaryote homologs ( 18).
However, uncertainties in the alignment of GABA AR subunit sequences
relative to those of the nAChR result in alternative GABA AR
homology models ( 12,
19,
20) that differ in the
location of amino acids in the M3 and M4 membrane-spanning helices and in the
M1 helix in some models ( 16,
21).If etomidate and neurosteroids both bind at the same β/α
interface in the GABA AR transmembrane domain, the limited space
available for ligand binding suggests that their binding pockets might overlap
and that ligand binding would be mutually exclusive. To address this question,
we photolabeled purified bovine brain GABA AR with
[ 3H]azietomidate in the presence of different neuroactive steroids
and determined by protein microsequencing whether active neurosteroids
inhibited labeling of α1Met-236 and βMet-286, as expected for
mutually exclusive binding, or resulted in [ 3H]azietomidate
photolabeling of other amino acids, a possible consequence of allosteric
interactions. Active steroids failed to inhibit labeling and enhanced labeling
of α1Met-236, clearly indicating that the neurosteroid and the etomidate
sites are distinct. Our GABA AR homology model that positions
α1Met-236 and βMet-286 at the β/α interface, but not
that of Hosie et al.
( 16), is also consistent with
cysteine substitution cross-linking studies
( 20,
22), which define the
proximity relations between amino acids in the αM1, αM2,
αM3, and βM3 helices, and these results support the interpretation
that the two residues photolabeled by [ 3H]azietomidate are part of
a single subunit interface binding pocket, whereas the steroid sensitivity
determinants identified by mutagenesis neither are at the β/α
subunit interface nor are contributors to a common binding pocket. 相似文献
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