Divorce of obligatory partners in pain: disruption of GABA(B) receptor heterodimers in neuralgia

EMBO J 31: 3239–3251 (2012); published online June122012It is now well established that G protein-coupled receptors can exist not only as homodimers, but also as heterodimers or higher order oligomers. However, whether and how dimerization of the receptors is regulated is poorly understood. In this issue of The EMBO Journal, the team of Marc Landry provides evidence for an intriguing mechanism by which—under pathological conditions—GABA_B receptor heterodimers at the cell surface are disrupted and thereby inactivated. An impressive set of experiments thus reveals a novel mechanism regulating the number of functional GABA_B receptors in the plasma membrane and shows that the receptor heterodimer may not be as stable as we previously thought.It is evident that dimerization and oligomerization at least of class A and C G protein-coupled receptors (GPCRs) play important roles in permitting or enhancing their cell surface trafficking (Milligan, 2010). The assembly process is thought to serve as a quality control mechanism to ensure that only fully mature and functional receptors reach the plasma membrane. The prototype of an obligatory heterodimer among GPCRs is the GABA_B receptor, which controls excitability of neurons by mediating slow inhibitory neurotransmission (Gassmann and Bettler, 2012). Functional GABA_B receptors are built from two related proteins termed as GABA_B1 and GABA_B2. Although both subunits display a similar structural organization—with a large extracellular domain containing a Venus fly-trap structure, seven transmembrane domains and a large intracellular located C-terminal domain—they serve distinct, complementary functions. GABA_B1 binds the orthosteric ligands whereas GABA_B2 recruits the G protein and is required for cell surface trafficking of the receptor complex by masking an ER retention signal present in the C-terminal domain of GABA_B1. This is a striking example that dimerization is essential for and control of expression of a functional GPCR. The availability of GABA_B receptors at the cell surface is also determined by receptor trafficking, which includes endocytosis, recycling and degradation of the receptors (Benke, 2010). To maintain the required number of cell surface receptors for signalling under a given physiological status, all levels of receptor trafficking need to be precisely balanced. Changing the balance of the different trafficking mechanisms is one means to adjust receptor numbers to changing physiological conditions. An example of such regulation is the recently uncovered mechanism of the glutamate receptor-mediated downregulation of GABA_B receptors where the balance of recycling and degradation of the receptors is shifted towards degradation (Benke et al, 2012). Laffray et al (2012) propose a novel and unexpected mechanism regulating the number of functional receptors at the cell surface. This mechanism is operative in vivo under pathological conditions and is based on the disruption of GABA_B receptor heterodimers present in the plasma membrane by the GABA_B1 interacting protein 14-3-3ζ.Seven members of 14-3-3 proteins (14-3-3β, γ, ɛ, ζ, η, σ and τ) are ubiquitously expressed in mammals. 14-3-3 proteins bind predominantly to phosphoserine and phosphothreonine containing sequences and interact with hundreds of different partners to regulate a variety of cellular processes ranging from protein trafficking, apoptosis, cell cycle, signal transduction, cell adhesion and metabolism. It is therefore not surprising that alterations in the expression levels of 14-3-3 proteins and/or changes in the interaction status with target proteins are increasingly observed in diseases such as cancer, neurodegenerative diseases and epilepsy (Zhao et al, 2011).Among 14-3-3 proteins, 14-3-3ζ interacts with the C-terminal domain of GABA_B1 and has been shown in vitro to inhibit the heterodimerization of GABA_B1 and GABA_B2 C-terminal domains (Couve et al, 2001). However, the physiological and potential pathological function of this interaction was entirely unresolved. In a rat model of neuropathic pain (spinal nerve ligation), Laffray et al (2012) observed a significant upregulation of 14-3-3ζ selectively in the ipsilateral dorsal horn of the lumbar spinal cord, the area where nociceptive signal processing in response to the injury takes place. Using several complementary methodologies including coimmunoprecipitation, colocalization immunofluorescence analysis, electron microscopy and two-photon fluorescence lifetime imaging, the authors demonstrated in vitro and in vivo that upregulation of 14-3-3ζ results in an increased interaction with GABA_B1 in the plasma membrane and in a concomitant loss of GABA_B1/GABA_B2 association. This finding suggests that 14-3-3ζ disrupts existing heterodimers in the plasma membrane (Figure 1). As a consequence, the increased GABA_B1/14-3-3ζ interaction rendered cell surface GABA_B receptors non-functional and impaired GABA_B receptor signalling.Open in a separate windowFigure 1Novel mechanism regulating GABA_B receptor signalling by disrupting the functional receptor heterodimer via interaction with 14-3-3ζ. Functional GABA_B receptors are obligatory heterodimers built from GABA_B1 and GABA_B2 subunits. Under normal conditions, binding of GABA to the Venus fly trap-like structure in the N-terminal domain of GABA_B1 activates Gi/o proteins recruited by GABA_B2 and thereby modulates distinct effector systems (adenylyl cyclases, potassium channels and voltage-gated Ca²⁺ channels). After induction of neuropathic pain by spinal nerve ligation, 14-3-3ζ is selectively upregulated in the spinal dorsal horn where painful sensory signals are processed and transmitted to the brain. 14-3-3ζ binds to the C-terminal domain of GABA_B1 and disrupts by a yet-to-be identified mechanism the receptor dimer. This results in non-fuctional receptors and prevents GABA_B receptor signalling.The main unresolved and extremely interesting issue concerns the mechanism of heterodimer disruption by 14-3-3ζ. The interaction site of 14-3-3ζ partially overlaps with the coiled-coil domain in the C-terminal domain of GABA_B1 (Couve et al, 2001). Coiled-coil domains are protein–protein interaction sites and are one of the domains thought to be involved in the heterodimerization of GABA_B1 and GABA_B2. The most obvious mechanism is a direct competition of 14-3-3ζ and GABA_B2 for interaction with GABA_B1. 14-3-3 proteins are inherently rigid proteins able to stabilize a given conformation after binding to its partner protein (Obsil and Obsilova, 2011). Thus, binding of 14-3-3ζ might arrest GABA_B1 in a conformation that is non-permissive for GABA_B2 heterodimerization. However, the apparent affinity of the interaction of 14-3-3ζ with GABA_B1 is rather low and relatively high concentrations of 14-3-3ζ are required to prevent heterodimerization of GABA_B1 and GABA_B2 C-terminal domains in vitro (Couve et al, 2001). Given the relatively moderate increase of 14-3-3ζ in neuropathic spinal cord, a direct competition mechanism per se appears unlikely. On the other hand, 14-3-3 proteins predominantly bind to motifs containing phosphorserine and phosphothreonine. Therefore, phosphorylation of GABA_B1 within the 14-3-3ζ binding site might thus foster the GABA_B1/14-3-3ζ interaction. In this regard, it would be important to test whether serine or threonine residues within the 14-3-3ζ binding site are phosphorylated in chronic pain states and whether phosphorylation is required for 14-3-3ζ interaction.An alternative mechanism may be based on the scaffolding properties of 14-3-3 proteins. 14-3-3 proteins act as dimers and thus harbour at least two protein interaction sites (Obsil and Obsilova, 2011). Therefore, 14-3-3ζ may target a second protein or a protein complex to GABA_B receptors that forces the receptor complex to dissociate and prevent reassociation. Proteomic analyses of isolated GABA_B1/14-3-3ζ complexes are needed to address this issue.Another important aspect of the paper is that it sheds some light on the involvement of GABA_B receptors in neuropathic pain. So far, there is no coherent picture on the contribution of GABA_B receptors to chronic pain states. However, there is increasing evidence that diminished GABA_B receptor activity due to downregulation of the receptors might play a role in at least some models of neuropathic pain (Zeilhofer et al, 2012). Although there might be distinct mechanisms downregulating functional GABA_B receptors in chronic pain conditions, disruption of GABA_B receptor heterodimers via upregulation of 14-3-3ζ appears to be a contributing factor. In their neuropathic pain model, Laffray et al (2012) observed a diminished analgesic activity of the intrathecally injected GABA_B receptor agonist baclofen. Preventing the binding of 14-3-3ζ to GABA_B receptors via knocking-down 14-3-3ζ with siRNA or by using a synthetic peptide disrupting the GABA_B1/14-3-3 interaction restored expression of GABA_B receptor heterodimers in the plasma membrane and consequently enhanced the analgesic effect of baclofen. Even more important, disruption of the 14-3-3ζ/GABA_B1 interaction by injection of the interfering synthetic peptide alone in the absence of baclofen partially reversed pain in the neuropathic rats. This finding implies that diminished GABA_B receptor signalling contributes to the expression of neuropathic pain. These results might be a starting point for a therapeutic strategy to reduce neuropathic pain based on reversing the GABA_B1/14-3-3ζ interaction. There are already small molecule inhibitors of 14-3-3 protein–protein interactions under development (Zhao et al, 2011), which might be useful for testing the feasibility of such an approach.     

Altered patterns ofN-linked glycosylation of theTorpedo acetylcholine receptor expressed inXenopus oocytes

Summary The nicotinic acetylcholine receptor (AChR) fromTorpedo electroplax is an oligomeric transmembrane glycoprotein made up of four highly homologous subunits in a stoichiometry of ₂. The role ofN-linked glycosylation of the AChR has been studied in several cell lines and these studies have suggested that the addition of carbohydrate may be important for receptor expression. WhileXenopus oocytes have proven to be an invaluable tool for studying the AChR, little is known aboutN-linked glycosylation of the oocyte-expressed receptor. The present report demonstrates that the oocyte-expressed AChR is glycosylated and contains the same number of oligosaccharide residues per subunit as the native receptor. However, unlike the nativeTorpedo receptor which contains both high mannose and complex oligosaccharides, the oocyte-expressed AChR contains only high mannose oligosaccharide modifications. However, as has been well documented, theTorpedo AChR expressed in oocytes is fully functional, demonstrating that the precise nature of the oligosaccharide modification is not critical for receptor function.The role of the oligosaccharide component of the AChR in receptor function was examined using tunicamycin (TM) to inhibitN-linked protein glycosylation. TM treatment resulted in a 70–80% inhibition of AChR expression in oocytes. Functional, unglycosylated receptors were not expressed; receptors expressed in TM-treated oocytes were functional wild-type, glycosylated AChR, formed only during the initial 12 hr of TM exposure. These data suggest that while glycosylation of the oocyte-expressedTorpedo AChR is required for assembly of subunits into a functional receptor, as has been demonstrated in other cells, oocyte modification of normalTorpedo glycosylation patterns does not affect receptor function or assembly.     

Determinants of Phencyclidine Potency on the Nicotinic Acetylcholine Receptors from Muscle and Electric Organ

1.Phencyclidine (PCP) is an inhibitor of the nicotinic acetylcholine receptor (AChR) with characteristics of an open-channel blocker. The location of PCP binding site on the AChR molecule is unknown.2.PCP inhibits the AChR from electric organ with a higher potency than muscle AChR. To find the molecular basis of this difference, we expressed the two native and six hybrid receptors, and two receptors containing mutated mouse subunits in Xenopus laevis oocytes. The inhibition of ACh-induced current in these receptors by PCP was studied using whole-cell voltage-clamp. All hybrid receptors generated robust ACh-induced currents, while incomplete receptors (-less or -less) did not.3.PCP potency was higher on hybrids containing Torpedo and subunits regardless of the and subunit origin. A mouse subunit containing the asparagine 6 to the serine mutation in the M2 segment conferred a high sensitivity to PCP.4.These results support the conclusion that the amino acid residues at the position 6 of the M2 segments contribute to the PCP potency difference between Torpedo and mouse receptors.5.Another noncompetitive inhibitor of the AChR, the cembranoid eupalmerin acetate (EUAC), also inhibited the electric organ receptor with a somewhat higher potency than muscle AChR. However, the IC₅₀ values for EUAC inhibition of hybrid receptors did not follow the pattern observed for PCP. Therefore, these two inhibitors interact differently with the AChR molecule.     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.     

Clozapine's Antipsychotic Effects do not Depend on Blockade of 5-HT3 Receptors

Sixteen known 5-HT₃ receptor blockers, including clozapine, fully or partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, indicating that they are also GABA_A antagonists, some of them selective for subsets of GABA_A receptors. The 5-HT₃ receptor blocker, ondansetron, has been reported to produce some antipsychotic and anxiolytic effects. However, no antipsychotic effects have been reported for a large number of highly potent 5-HT₃ receptor blockers. Like clozapine, ondansetron partially reverses the inhibitory effect of GABA on [³⁵S]TBPS binding. Additivity experiments suggest that ten 5-HT₃ receptor blockers tested at low concentrations preferentially block subtypes of GABA_A receptors that are among those blocked by clozapine. Wiley and Porter (29) reported that MDL-72222, the most potent GABA_A antagonist decribed here, partially generalizes (71%) with clozapine in rats trained to discriminate an interoceptive clozapine stimulus, but only at a dose that severly decreases responding. Tropisetron (ICS-205,930) exhibits both GABA-positive and GABA-negative effects. R-(+)-zacopride is 6-fold more potent than S-(–)-zacopride as a GABA_A antagonist. We conclude that the observed antipsychotic and, possibly, anxiolytic effects of some 5-HT₃ receptor blockers are due to selective antagonism of certain GABA_A receptors, and not to blockade of 5-HT₃ receptors. We speculate that the anxiolytic and sedative effects of clozapine and several other antipsychotic drugs may be due to selective blockade of 122 GABA_A receptors which are preferentially located on certain types of GABAergic interneurons (probably parvalbumin positive). Blockade of these receptors will increase the inhibitory output of these interneurons. So far, no highly potent GABA_A antagonists with clozapine-like selectivity have been identified. Such compounds may exhibit improved clozapine-like antipsychotic activity.     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Assembly,trafficking and function of α1β2γ2 GABAA receptors are regulated by N‐terminal regions,in a subunit‐specific manner

GABA_A receptors are pentameric ligand‐gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand‐gated ion channels structures, sequence analysis predicts an α‐helix near the N‐terminus of each GABA_A receptor subunit. Preceding each α‐helix are 8–36 additional residues, which we term the N‐terminal extension. In homomeric GABA_C receptors and nicotinic acetylcholine receptors, the N‐terminal α‐helix is functionally essential. Here, we determined the role of the N‐terminal extension and putative α‐helix in heteromeric α1β2γ2 GABA_A receptors. This role was most prominent in the α1 subunit, with deletion of the N‐terminal extension or further deletion of the putative α‐helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N‐terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α‐helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α‐helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N‐terminal extensions and α‐helices make key subunit‐specific contributions to assembly, consistent with both regions being involved in inter‐subunit interactions.

     

Amphiporus alkaloid multiplicity implies functional diversity: initial studies on crustacean pyridyl receptors

Some hoplonemertines contain pyridyl alkaloids which are used for prey capture and chemical defense against predators. The first such alkaloid to be identified was anabaseine, a tetrahydro-bipyridyl isolated from the peregrine hoplonemertine Paranemertes peregrina Coe. Several Amphiporus species have also been shown to possess related pyridyl alkaloids. The chevron hoplonemertine Amphiporus angulatus (Fabricius) contains a particularly diverse group of bipyridyl and tetrapyridyl compounds. 2,3-bipyridyl, a fully aromatic analog of anabaseine, is the major paralytic toxin (Kem et al., 1976). It is even more potent in paralyzing crustaceans than anabaseine, but appears to be inactive on vertebrates. We have synthesized a number of 2,3-bipyridyl analogs and tested their ability to paralyze the freshwater crayfish Procambarus clarkii Girard. A structure-activity relationship (SAR) was found that resembles the SAR for activation of vertebrate nicotinic receptors by methylated nicotine and anabaseine compounds. This suggests that 2,3-bipyridyl, though lacking a positive charge at physiological pH, still acts as a nicotinic agonist in crustaceans. 2,3-bipyridyl and anabaseine stimulate crustacean dactyl neuron pyridyl chemoreceptors at M concentrations. Nemertelline, as well as these two compounds, was found to activate crayfish stomach muscle nicotinic receptors modulating chloride channels at sub-micomolar concentrations. We suggest that some amphiporid pyridyl alkaloids may deter predators not only by their central neurotoxic (paralytic) actions, but also by peripheral effects on pyridyl receptors affecting prey capture and digestion. A multiplicity of alkaloids probably provides the hoplonemertine with a variety of offensive and defensive chemical responses to the presence of other animals.     

GABA-gated anion channels in intact crayfish opener muscle fibres and stretch-receptor neurons are neither activated nor desensitized by glutamate

Summary The influence of glutamate on the GABA-activated Cl^- conductance was studied in the slowly adapting stretch-receptor neuron and dactylopodite opener muscle fibre of the crayfish (Astacus astacus) using a two-microelectrode and a three-microelectrode voltage clamp, respectively. Glutamate (0.5–1.0 mM) had no effect on the GABA-activated conductance in either preparation. This indicates that the availability of the inhibitory channels for activation of GABA is not influenced by glutamate. The present results are in sharp contrast to those obtained by Franke et al. (J Comp Physiol A 159:591–609, 1986) in experiments on excised membrane patches, which suggested that glutamate is capable of both activating and desensitizing inhibitory postsynaptic channels in the crayfish opener muscle fibre.Abbreviations GABA -aminobutyric acid - G_GABA and G _GABA ^p GABA-gated conductance and peak conductance - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - I current - SRN stretch-receptor neuron - V_m and V_l membrane voltage in two- and three-microelectrode voltage clamp, respectively     

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.     

The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

Abstract

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.     

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to ��-Aminobutyric Acid Type A Receptors

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.     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

Possible Involvement of GABAA and GABAB Receptors in the Inhibitory Action of Lindane on Transmitter Release from Cerebellar Granule Neurons

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA_A and GABA_B classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 M THIP as this is known to influence the properties of both GABA_A and GABA_B receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K⁺) release of neurotransmitter ([³H]D-aspartate as label for glutamate). In naive cells both GABA_A and GABA_B receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA_A and GABA_B receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA_A and GABA_B receptors is able to indirectly interfere with both GABA_A and GABA_B receptors. In case of the latter receptors it was shown using [³H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.     

Clozapine and Several Other Antipsychotic/Antidepressant Drugs Preferentially Block the Same ‘Core’ Fraction of GABAA Receptors

Clozapine and several other antipsychotic/antidepressant drugs that fully or partially block GABA_A receptors were tested at concentrations that reversed the inhibitory effect of 1 M GABA on ³⁵S-t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding to rat forebrain membranes only about 20–30%, here designated core fractions. Clozapine at 10 M reverses 1 M GABA 25 ± 4.0% (n = 23) (its core fraction). Fourty three compounds were tested alone, and pairwise together with 10 M Clozapine. The core fractions of some of the compounds yielded significant additive reversals together with 10 M Clozapine, while others did not. A group of 14 compounds of which 7 are clinically effective antipsychotic drugs, including Chlorprothixene, Clomacran, Clopipazan, Fluotracen, Sulforidazine, Thioproperazine, and cis-Thiothixene, were statistically non-additive with 10 M Clozapine, suggesting that all of these drugs selectively block the same core population of GABA_A receptors as Clozapine. These non-additivities also suggest that Clozapine at 10 M fully saturates a subset of GABA_A receptors blocked by 1 M GABA. Therefore, Clozapine probably blocks 2 or more types of GABA_A receptors, but only half of the receptors that are sensitive to 1 M GABA. A second group of 12 compounds of which 6 are clinically active antidepressant/antipsychotic drugs including Amoxapine, Clothiapine, Dibenzepine, Inkasan (Metralindole), Metiapine and Zimelidine were slightly, but significantly, additive with Clozapine suggesting that these compounds block most of Clozapine's core fraction, plus a small additional fraction. A third group consisted of ten compounds that yielded larger (R > 80) and statistically highly significant additivities with Clozapine. Complete additivity was obtained with Bathophenanthroline disulfonate, and Isocarboxazid, suggesting that they block GABA_A receptors other than those blocked by 10 M Clozapine. Seven classical GABA_A receptor blockers, also tested at concentrations yielding 21 to 33% reversal alone, were all significantly additive with 10 M Clozapine, but in no case was the additivity complete. The largest additivity was obtained with Pitrazepine (21%) and the smallest with Tubocurarine (9%). These results provide further support for the notion that selective blockade of the same subset of GABA_A receptors may contribute to the clinical antipsychotic/antidepressant effects of Clozapine. The B_opt values for Clozapine are 50 ± 1.7% and 26 ± 2.6% ( n = 3) in whole rat forebrain and cerebellum, respectively, confirming that clozapine-sensitive GABA_A receptors are unevenly distributed in the brain. The sedative and anxiolytic properties of Clozapine and other antipsychotic drugs may be due to selective blockade of GABergic disinhibition at certain interneurons.     

Endogenous Benzodiazepine Site Peptide Ligands Operating Bidirectionally In Vivo in Neurogenesis and Thalamic Oscillations

By binding to the benzodiazepine site, diazepam binding inhibitor (DBI) is associated with negative allosteric modulation (NAM) of GABA_A receptors (Costa and Guidotti in Life Sci 49:325–344, 1991). However, the demonstration of a true physiological role of DBI and its fragments has only recently been reported. Based on DBI gain- and loss-of-function experiments in vivo, DBI and its fragment ODN were found to promote neurogenesis in the subventricular zone in vivo. Acting as NAM on GABA_A receptors of precursor cells, DBI counteracted the inhibitory effect of GABA and thereby enhanced the proliferation of these cells (Alfonso et al. in Cell Stem Cell 10:76–87, 2012). Conversely and most remarkably, in similar gain- and loss-of-function experiments in the thalamus, the DBI gene products acted as positive allosteric modulators (PAM) of GABA_A receptors in prolonging the duration of IPSCs, an effect which was specific for GABA transmission within the reticular nucleus (nRT) (Christian et al. in Neuron 78:1063–1074, 2013). Since intra-nRT potentiation of GABA transmission by benzodiazepine drugs exerts powerful anti-oscillatory effects, DBI might be endogenously effective by modulating seizure susceptibility. It remains to be seen by which mechanism both NAM and PAM activity can arise from the Dbi gene. Nevertheless, the results open new perspectives on the regionally distinct endogenous modulation of GABA transmission via the benzodiazepine site.     

Does gabapentin act as an agonist at native GABA<Subscript>B</Subscript> receptors?

Gabapentin, a novel anticonvulsant and analgesic, is a -aminobutyric acid (GABA) analogue but was shown initially to have little affinity at GABA_A or GABA_B receptors. It was recently reported to be a selective agonist at GABA_B receptors containing GABA_B1a-GABA_B2 heterodimers, although several subsequent studies disproved that conclusion. In the present study, we examined whether gabapentin is an agonist at native GABA_B receptors using a rat model of postoperative pain in vivo and periaqueductal gray (PAG) slices in vitro; PAG contains GABA_B receptors, and their activation results in antinociception. An intrathecal injection of gabapentin or baclofen, a GABA_B receptor agonist, induced antiallodynia in this postoperative pain model. Intrathecal injection of GABA_B receptor antagonists CGP 35348 and CGP 55845 antagonized baclofen- but not gabapentin-induced antiallodynia. In ventrolateral PAG neurons, baclofen activated G-protein-coupled inwardly rectifying K⁺ (GIRK) channels in a manner blocked by CGP 35348 or CGP 55845. However, gabapentin displayed no effect on the membrane current. In neurons unaffected by gabapentin, baclofen activated GIRK channels through GABA_B receptors. It is concluded that gabapentin is not an agonist at GABA_B receptors that are functional in baclofeninduced antiallodynia in the postoperative pain model in vivo and in GIRK channel activation in ventrolateral PAG neurons in vitro.