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    GABA-Benzodiazepine Receptor Complex

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    Explain in detail the GABA-Benzodiazepine Receptor Complex. What are the effects of benzodiazepines, barbiturates, and alcohol on this complex?

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    Interesting questions, indeed. Please see response below, some of which is attached. I hope this helps and take care.


    1. Explain in detail the GABA-Benzodiazepine Receptor Complex. What are the effects of benzodiazepines, barbiturates, and alcohol on this complex?

    Our understanding of the function and pharmacology of the GABA-benzodiazepine receptor is currently undergoing a major revolution through the use of knockout and knock-in techniques and the discovery of drugs selective for particular subtypes of the GABA-benzodiazepine receptor. The GABA-benzodiazepine receptor comprises five protein subunits drawn from several different families (e.g., alpha1-6, beta1-3, gamma, delta, and rho) that come together to form an ion channel with binding sites or receptors for the endogenous transmitter GABA, benzodiazepines, and other agents such as neurosteroids and convulsants (Sieghart, 1995). When GABA binds with the GABA-benzodiazepine receptor complex, conformational changes increase the permeability of the central pore to chloride ions (Nutt and Malizia, 2001). The resulting chloride flux hyperpolarizes the neuron, reducing its excitability and producing a general inhibitory effect on neuronal activity. Other compounds, such as barbiturates and neurosteroids, also bind to this receptor complex to increase channel opening. Benzodiazepines, such as diazepam, also increase channel opening but do so through increasing the effectiveness of GABA rather than by directly opening the chloride channel. Moreover, because the alpha5-containing GABA-benzodiazepine receptors are predominantly located in the limbic system, this suggests they play a role in emotional states, memory, and in disease processes such as dementia and anxiety (see more detail in the article attached at the end of this response, which is also available on-line at http://www.nature.com/jcbfm/journal/v22/n7/full/9591280a.html).

    GABA is formed in the brain from glutamate, glucose, and glutamine, and binds to one of two receptors on the postsynaptic neuron. GABA A receptors regulate excitability and anxiety, panic, and stress, and are the targets of benzodiazepines such as Ativan, barbiturates, and alcohol. Depressed individuals have decreased GABA in their cerebral spinal fluid and plasma. (http://www.mcmanweb.com/article-236.htm).

    Specifically, the GABA A receptor is one of a family of neurotransmitter receptors with fast intrinsic ion channels that includes the glycine receptor and the nicotinic acetylcholine receptor. Distinct from another major receptor family, the muscarininc acetylcholine receptor and rhodopsin, with no intrinsic ion channel. The A receptor is specifically blocked by bicuculline. It consists of two pairs of protein chains forming an A2B2 complex, the A chains bind benzodiazepine and the B chains bind GABA. The 4 subunits are thought to form a tight group with the chloride channel in the middle. There is considerable similarity between the amino acid sequences of the receptor subunits and those of the nicotinic acetylcholine receptor suggesting that both receptors are derived from some evolutionary ancestor.


    BENZODIAZEPINE receptors in the brain (1) are activated by γ-aminobutyric acid (GABA) and by the GABA agonist muscimol in vitro (2). This activation may be related to the GABA-potentiating effects of benzodiazepines observed in electro-physiological studies (see ref. 3 for review). The enhancement of specific 3H-diazepam binding by GABA agonists is inhibited by bicuculline and thus offers a unique high-affinity binding system for investigations in vitro of agonist-antagonist interactions at GABA receptors in the central nervous system. We report here that two GABA-mimetic compounds, 3-aminopropane-sulphonic acid (APS) and isoguvacine, are partial agonists (mixed agonists/antagonists) with intermediate efficacies. Imidazoleacetic acid (IAA) is also a partial agonist but with only marginal agonist activity, and the new GABA-mimetics piperidine-4-sulphonic acid (PSA) and 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP) are competitive antagonists to the GABA/benzodiazepine receptor complex (http://adsabs.harvard.edu/abs/1979Natur.280..331B)

    EXAMPLE: The GABA/benzodiazepine receptor complex in the nervous system of a hypertensive strain of rat
    (Tunnicliff G, Welborn KL, Head RA)

    Neurochem Res 1984 Aug;9(8):1033-8.

    gamma-Aminobutyric acid (GABA) has been implicated in the development of hypertension and in the regulation of blood pressure. The spontaneously hypertensive rat (SHR) offers an opportunity to explore the role of central GABA and other neurotransmitters in the genesis of high blood pressure. The receptor binding of [3H]GABA, [3H]flunitrazepam, and [3H]glutamate to synaptic membranes from the cerebral cortex and cerebellum of SHR rats were measured in animals of various ages. No significant differences between the SHR and a normotensive control strain of rats were found for any of the assays. The results indicate that in this model of hypertension, neither GABA nor glutamate function are involved, at least not in the cerebral cortex or cerebellum.

    B. What are the effects of benzodiazepines, barbiturates, and alcohol on this complex?

    Receptor sites for drug and neurotransmitter binding are associated with the GABA receptor complex, which serves as a primary site of action of benzodiazepines, barbiturates and other sedative-hypnotics, such as alcohol.6 Benzodiazepines and barbiturates act at separate binding sites on the receptor to potentiate the inhibitory action of GABA. They do so by allosterically altering the receptor (changing its conformation) so that it has a greater binding affinity for GABA. Ethanol modifies the receptor by altering the membrane environment so that it has increased affinity for GABA and the other sedative-hypnotic drugs. That benzodiazepines, barbiturates and ethanol all have related actions on a common receptor type, which explains their pharmacologic synergy and cross tolerance. Thus, benzodiazepines are used during alcohol detoxification.

    a. Ethanol has a broad range of actions on many neurotransmitter systems. The depressant actions of ethanol in the brain are related in part to facilitation of [gamma]-aminobutyric acid (GABA) neurotransmission via its interaction with the benzodiazepine/GABA receptor complex. As mentioned above, ethanol modifies the receptor by altering the membrane environment so that it has increased affinity for GABA and the other sedative-hypnotic drugs.

    EXAMPLE: Ethanol and the benzodiazepine-GABA receptor-ionophore complex (Ticku MK)

    University of Texas Health Science Center, Department of Pharmacology, San Antonio 78284-7764.

    Ethanol has a pharmacological profile similar to that of classes of drugs like benzodiazepines and barbiturates, which enhance GABAergic transmission in the mammalian CNS. Several lines of behavioral, electrophysiological and biochemical studies suggest that ethanol may bring about most of its effects by enhancing GABAergic transmission. Recently, ethanol at relevant pharmacological concentrations has been shown to enhance GABA-induced 36Cl-fluxes in cultured spinal cord neurons, synaptoneurosomes and microsacs. These enhancing effects of ethanol were blocked by GABA antagonists. Ro15-4513, an azido analogue of classical BZ antagonist Ro15-1788, reversed most of the behavioral effects of ethanol and other effects involving 36Cl-flux studies. The studies summarized below indicate that most of the pharmacological effects of ethanol can be related to its effects on GABAergic transmission (http://www.arclab.org/medlineupdates/abstract_6149479.html).

    b. Benzodiazepines and Barbiturates

    Benzodiazepines: A class of drug widely used in medical practice as CNS depressants, for example diazepam (the tranquilliser Valium).

    Barbiturates : A class of chemicals derived from barbituric acid or thiobarbituric acid. Many of these are medically important as sedatives and hypnotics (sedatives, barbiturate), as anaesthetics, or as anticonvulsants.

    As mentioned above, both benzodiazepines and barbiturates act at separate binding sites on the receptor to potentiate the inhibitory action of GABA. They do so by allosterically altering the receptor (changing its conformation) so that it has a greater binding affinity for GABA. As mentioned above, instead, ethanol modifies the receptor by altering the membrane environment so that it has increased affinity for GABA and the other sedative-hypnotic drugs.

    With long-term high-dose use of benzodiazepines (or ethanol), there is an apparent decrease in the efficacy of GABA-A receptors, presumably a mechanism of tolerance.6,7 When high-dose benzodiazepines or ethanol are abruptly discontinued, this "down-regulated" state of inhibitory transmission is unmasked, leading to characteristic withdrawal symptoms such as anxiety, insomnia, autonomic hyperactivity and, possibly, seizures (see http://www.aafp.org/afp/20000401/2121.html).

    EXAMPLE: Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex
    (Stephenson FA, Olsen RW)

    Barbiturates enhance the binding of [3H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, KD, for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [3H]Flunitrazepam binding activity is stable with storage at 4 degrees C, but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly (t 1/2 = 48 h). [3H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6292364&dopt=Abstract).


    EXTRA READING: (From http://www.nature.com/jcbfm/journal/v22/n7/full/9591280a.html, click to see diagram and tables).

    There are profound differences in the distribution of the common receptor subunits in the brain, the alpha1 being most abundant, particularly in the cortex (Fritschy and Möhler, 1995; Pirker et al., 2000; Wisden et al., 1992). In contrast, the alpha2 and alpha5 types have a more limited distribution and predominate in the hippocampus. The thalamus is rich in alpha4 subunits, whereas the alpha6 subunit is almost exclusively found in cerebellum (Fritschy and Möhler et al., 1995; Pirker et al., 2000; Wisden et al., 1992). Such a distribution suggests that different subtypes may mediate different functions, and recently it has been proposed that the sedative but not anxiolytic effects of diazepam are mediated through the alpha1 subtype (Rudolph et al., 1999).

    Given this definition of subtype functions in animal models, it is timely to conduct such characterization in humans. Benzodiazepine receptor function in humans can be assessed in vivo by measuring the effects of a benzodiazepine on sedation, anxiety, EEG, or saccadic eye movements (Roy-Byrne et al., 1993). However, the effects on a particular anatomical or subtype population of GABA-benzodiazepine receptors cannot be determined by any of these methods. Both single photon emission computed tomography and positron emission tomography (PET) have been used extensively to delineate the distribution and levels of the GABA-benzodiazepine receptor in a variety of neuropsychiatric disorders including anxiety, alcoholism, and epilepsy (Malizia and Richardson, 1995).

    The commonly used radioligands for PET and single photon emission computed tomography are [11C]flumazenil and [123I]iomazenil (iodinated derivative of flumazenil), respectively. Flumazenil is a benzodiazepine antagonist that binds with high affinity to benzodiazepine receptors containing the alpha1, alpha2, alpha3, or alpha5, (Ki approximately 1 nmol/L) subunits and less so to those containing alpha4 or alpha6 subunits (Ki approximately 150 nmol/L) (Sieghart, 1995). Because of the high abundance of alpha1-containing receptors in the brain, a PET or single photon emission computed tomography image of benzodiazepine receptor binding appears to primarily reflect this population. Although other radioligands have been investigated (suriclone, flunitrazepam, triazolam), none has proved as useful, and also label a similar population of GABA-benzodiazepine receptors, as [11C]flumazenil or [123I]iomazenil (Pike et al., 1993). More recently, other radioligands?[3H]L655,708 and [3H]RY80?have been evaluated as putative PET ligands of the alpha5-containing GABA-benzodiazepine receptor, but were not sufficiently retained in the brain (Opacka-Juffry et al., 1999). Consequently, there is a paucity of probes for imaging selectively in vivo GABA-benzodiazepine receptor subtypes.

    Ro15 4513 is a partial inverse agonist at the GABA-benzodiazepine receptor. [11C]Ro15 4513 was developed as a potential PET radioligand (Halldin et al., 1992; Inoue et al., 1992; Sazdot et al., 1989). Early studies revealed that [11C]Ro15 4513 bound with a very different regional brain profile to flumazenil, suggesting that it labeled a different population of GABA-benzodiazepine receptors (Halldin et al., 1992; Inoue et al., 1992; Onoe et al., 1996). Its use as a PET radiotracer has been limited because of the preeminence of [11C]flumazenil and the uncertainty about which GABA-benzodiazepine receptors were being labeled, though the alpha5-containing subtype was suggested but never demonstrated. In view of the recent knowledge concerning the subtypes of the GABA-benzodiazepine receptor and data suggesting that Ro15 4513 has relatively higher affinity for the alpha5-containing subtype (Hadingham et al., 1993), we decided to validate [11C]Ro15 4513 as a PET radioligand that could be used to characterize the alpha5 subtype in humans. We used our small animal PET study to characterize its binding in rats and tested its subtype selectivity by pretreating rats with compounds of known subtype selectivity, such as zolpidem (alpha1), flunitrazepam (alpha1, alpha2, alpha3, alpha5), RY80 (alpha5), and L655,708 (alpha5) (Hadingham et al., 1993; Quirk et al., 1996; Sieghart, 1995; Skolnick et al., 1997).


    [11C]Flumazenil was prepared with more than 95% radiochemical purity and with a specific radioactivity of approximately 24,000 MBq/mumol at the end of the synthesis by N-methylation of the corresponding N-desmethyl derivative with [11C]iodomethane, as described previously (Pike et al., 1993). [11C]Ro15 4513 was synthesized in a manner described by Inoue et al., (1992), by N-methylation of the corresponding N-desmethyl derivative with [11C]iodomethane. The product was purified by reverse phase HPLC on a column (Phenomenex Ultra-carb 7 ODS (30), 250 times 10-mm outer diameter) eluted with MeCN/0.03-mol/L MeCOONH4/AcOH (100/100/1 by volume). The specific radioactivity was approximately 14,000 MBq/mumol at end of synthesis.

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    Solution Summary

    This solution explains the GABA-Benzodiazepine Receptor Complex, including the effects of benzodiazepines, barbiturates, and alcohol on this complex. Illustrative examples provided.