Allosteric modulatory centers of transmitter amino acid receptors

Transmitter amino acid receptors (gamma-aminobutyric acid [GABA] and excitatory amino acids) include in their structure allosteric modulatory centers that regulate the probability of transmitter action. These are sites of action for drugs. In GABA receptors, benzodiazepines and beta-carbolines act as positive and negative modulators. Various subtypes of GABAA receptors exist that differ with regard to the structure of the receptor subunits and the characteristic of the allosteric modulatory centers. This brings up the possibility that classes of benzodiazepines exist that, by acting selectively on specific subtypes of GABAA receptors, may bring about selectivity of drug action in specific anxiety disorders. For instance, clonazepam appears to act better than diazepam on panic attacks and fails to bind to GABAA receptor subtypes located in spinal cord. Also, glutamate receptors and specifically the N-methyl-D-aspartate-sensitive subtype modulated by an allosteric center may include various molecular forms differing with respect to the properties of the allosteric modulatory center. This variability suggests that this center may be used as a target for discovery of drugs acting as specific allosteric modulators of glutamate receptors.     

Expression and regulation of GABA receptors in the brain]

GABA receptors are classified into two receptor subtypes: GABAA and GABAB receptors. The GABAA receptor, one of the ionotropic type receptors, is formed by various subunits (alpha, beta, gamma and delta subunits) and constitutes the GABA-gated Cl- channel. The different combinations of these subunits are known to produce functionally heterogeneous GABAA receptors both pharmacologically and physiologically. On the other hand, GABAB receptor is known to be metabotropic type which is negatively coupled with adenylate cyclase and inositol phosphate turnover systems via inhibitory GTP binding protein.     

Pharmacology of an omega receptor agonist]

It has been suggested that different BZP (omega) receptor subtypes may mediate distinct behavioral effects of BZP receptor ligands. Several studies demonstrated that omega1 selective compounds are characterized by an increase in slow wave deep sleep with rapid onset of hypnotic action, and by reduced muscle relaxation, amnesic liability or tolerance. On the other hand, it is known that many different subtypes of GABAA receptors exist, based on the fact that many different subtypes can go into assembling GABAA receptors. GABAA receptors with alpha1 subunits may mediate sedative action and perhaps amnesia. Those with alpha2 subunits probably mediate anxiolytic actions. GABAA receptors with alpha3 subunits may regulate various neurotransmitters, and those with alpha5 subunits may also contribute to amnesia. Such discoveries could open up new avenues for drug development.     

Distribution of GABAA and glycine receptors in the mammalian retina

1. GABA and glycine mediate synaptic inhibition via specific neurotransmitter receptors. Molecular cloning studies have shown that there is a great diversity of receptors for these two neurotransmitters. In the present paper, the distribution of GABAA and glycine receptors in the mammalian retina is reviewed. 2. In situ hybridization, immunocytochemistry with subunit-specific antibodies and single cell injection were used to analyse the localization of receptor subunits. Specific subunits are expressed in characteristic strata of the inner plexi-form layer, suggesting that different functional circuits involve specific subtypes of neurotransmitter receptors. 3. Different cell types express different combinations of receptor subunits and an individual neuron can express several receptor isoforms at distinct post-synaptic sites.     

Role of central GABAB receptors in physiology and pathology.

There are two major classes of gamma-aminobutyric acid (GABA)-sensitive receptors: GABAA and GABAB. The GABAA receptor, the better known of the two GABA receptors, is a heterooligomeric complex that forms a chloride channel. Multiple subtypes of the GABAA receptor result from the composition of different subunits. In contrast to the GABAA receptor, the GABAB receptor protein has not been isolated and purified to homogeneity. Various effector systems, however, have been identified for the GABAB receptor using a limited GABAB-specific pharmacologic reportoire. In almost all cases, activated GABAB receptors employ a guanosine triphosphate-binding protein to transduce a signal intracellularly. There may be multiple subtypes of the GABAB receptor. Because the responses elicited by activation of GABAB receptors are small in terms of their intensity and are considered to be modulatory, the role these receptors play in the central nervous system (CNS) may not be very obvious. However, it is our view that in a finely tuned instrument such as the brain, treatment with neuromodulators (drugs that produce slight changes in brain neurochemistry) may be safer than most current drugs. Moreover, neuromodulators may have far greater potential as pharmacotherapeutic agents for CNS disorders. Thus, in this article, we will review the pharmacologic characteristics of the GABAB receptor, known physiologic roles that this receptor plays in the CNS, and the importance of this receptor in certain disease states.     

Subunit composition,distribution and function of GABA(A) receptor subtypes

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. Depending on their subunit composition, these receptors exhibit distinct pharmacological and electrophysiological properties. Recent studies on recombinant and native GABA(A) receptors suggest the existence of far more receptor subtypes than previously assumed. Thus, receptors composed of one, two, three, four, or five different subunits might exist in the brain. Studies on the regional, cellular and subcellular distribution of GABA(A) receptor subunits, and on the co-localization of these subunits at the light and electron microscopic level for the first time provide information on the distribution of GABA(A) receptor subtypes in the brain. These studies will have to be complemented by electrophysiological and pharmacological studies on the respective recombinant and native receptors to finally identify the receptor subtypes present in the brain. The distinct cellular and subcellular location of individual receptor subtypes suggests that they exhibit specific functions in the brain that can be selectively modulated by subtype specific drugs. This conclusion is supported by the recent demonstration that different GABA(A) receptor subtypes mediate different effects of benzodiazepines. Together, these results should cause a revival of GABA(A) receptor research and strongly stimulate the development of drugs with a higher selectivity for alpha2-, alpha3-, or alpha5-subunit-containing receptor subtypes. Such drugs might exhibit quite selective clinical effects.     

γ-氨基丁酸A型受体在神经精神性疾病发生发展中的意义

γ-氨基丁酸（GABA）是中枢神经系统中介导抑制性突触传递的神经递质，通过GABAA、GABAB和GABAC三种亚型受体介导广泛的生理效应。GABAA受体亚型是GABA受体中占主导地位的亚型，可介导GABA的大部分功能，由来自8个亚基族（α，β，γ，δ，θ，ε，ρ和π）的不同亚基组成，而最典型的GABAA受体结构是由5个异质性多肽亚基（两个α、两个β和一个γ）组成的五边形寡聚体。不同亚基尤其是不同α亚基组成的亚型介导的生理和药理学效应有所不同。GABAB受体对焦虑、抑郁、癫痫以及记忆障碍等不同的神经精神性疾病的发生发展有重要的意义，可能是这些疾病防治药物的作用靶标。     

GABAA receptors: ligand-gated Cl- ion channels modulated by multiple drug-binding sites.

GABAA receptors are ligand-gated Cl- ion channels and the site of action of a variety of pharmacologically and clinically important drugs. In this review evidence is summarized indicating that these drugs, by interacting with several distinct binding sites at these receptors, allosterically modulate GABA-induced Cl- ion flux. Other results indicate that the affinity, as well as the modulatory efficacy of drugs, changes with receptor composition. A though investigation of the pharmacological properties of the individual binding sites on different GABAA receptor subtypes could open new avenues for selective modulation of GABAA receptors in different brain regions.     

GABA-based therapeutic approaches: GABAA receptor subtype functions

It is increasingly being appreciated that GABAA receptor subtypes, through their specific regional, cellular and subcellular localization, are linked to distinct neuronal circuits and consequently serve distinct functions. GABAA receptor subtype-selective drugs are therefore expected to provide novel pharmacological profiles. Receptors containing the alpha1 subunit mediate sedation and serve as targets for sedative hypnotics. Agonists selective for alpha2- and/or alpha3-containing GABAA receptors have been shown to provide anxiolysis without sedation in preclinical models, whereas inverse agonists selective for alpha5-containing GABAA receptors provide memory enhancement. Agonists selective for alpha3-containing GABAA receptors might be suitable for the treatment of deficits in sensorimotor processing in psychiatric disorders. Thus, a new pharmacology based on GABAA receptor subtype-specific actions is emerging.     

Regulation of gamma-aminobutyric acidA receptor subunit expression by activation of N-methyl-D-aspartate-selective glutamate receptors.

Exposure of primary cultures of rat cerebellar granule cells to specific antagonists of the N-methyl-D-aspartate (NMDA)-selective glutamate receptor reduces the steady state levels of mRNAs encoding various gamma-aminobutyric acidA (GABAA) receptor subunits. These neurons are glutamatergic and require a depolarizing concentration of K+ (25 mM) for optimal development and survival. When the neuronal differentiation rate is retarded by lowering of the extracellular [K+] (to 12.5 mM), a persistent stimulation of the same glutamate receptors with nonneurotoxic doses of NMDA increases the expression of these GABAA receptor subunits. This suggests that the lowered K+ concentration reduces neuronal depolarization and the consequent release of glutamate from the cells. These results show that the neuronal content of selected GABAA receptor subunit mRNAs is optimized by certain levels of glutamate in the culture medium, suggesting a neurotrophic action of this neurotransmitter at certain developmental stages of granule cells in culture.     

A novel GABA(A) receptor pharmacology: drugs interacting with the α(+) β(-) interface

GABA(A) receptors are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to a multiplicity of GABA(A) receptor subtypes with distinct subunit composition; regional, cellular and subcellular distribution; and pharmacology. Most of these receptors are composed of two α, two β and one γ2 subunits. GABA(A) receptors are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anaesthetics and convulsants. Whereas GABA acts at the two extracellular β(+) α(-) interfaces of GABA(A) receptors, the allosteric modulatory benzodiazepines interact with the extracellular α(+) γ2(-) interface. In contrast, barbiturates, neuroactive steroids and anaesthetics seem to interact with solvent accessible pockets in the transmembrane domain. Several benzodiazepine site ligands have been identified that selectively interact with GABA(A) receptor subtypes containing α2βγ2, α3βγ2 or α5βγ2 subunits. This indicates that the different α subunit types present in these receptors convey sufficient structural differences to the benzodiazepine binding site to allow specific interaction with certain benzodiazepine site ligands. Recently, a novel drug binding site was identified at the α(+) β(-) interface. This binding site is homologous to the benzodiazepine binding site at the α(+) γ2(-) interface and is thus also strongly influenced by the type of α subunit present in the receptor. Drugs interacting with this binding site cannot directly activate but only allosterically modulate GABA(A) receptors. The possible importance of such drugs addressing a spectrum of receptor subtypes completely different from that of benzodiazepines is discussed.     

The value of genetic and pharmacological approaches to understanding the complexities of GABA(A) receptor subtype functions: the anxiolytic effects of benzodiazepines

The identification of gamma-aminobutyric acid A (GABA(A)) receptor subunit genes over the last twenty years has shown that GABA(A) receptors are made up of many different subtypes. As such the dissection of which receptor subtypes mediate which functions of clinically useful GABAergic drugs, such as benzodiazepines, has been extremely complicated. Two complimentary approaches have been taken: the development of subtype-selective drugs and the genetic manipulation of different receptor subunits. Both have yielded exciting results, but sometimes with contradictory findings. This review highlights the strengths and weaknesses of both approaches, illustrating with specific discussion of the work, to uncover which receptor subtype(s) mediates the anxiolytic effects of benzodiazepines.     

Participation of GABAergic systems in the production of antinociception by various stresses in mice.

Based on the data that diazepam, a benzodiazepine (BZP) receptor agonist, antagonized psychological (PSY)-stress induced analgesia (SIA) without prominent action on footshock (FS)- and forced swimming (SW)-SIA and that BZP receptors are coupled with GABA receptors, we examined how the GABAergic system participates in the production of various SIAs. Muscimol, a GABAA receptor agonist, at doses of 0.25 to 1.0 mg/kg, affected each SIA differently, suppressed PSY-SIA at 0.25 mg/kg but tended to potentiate it at 1.0 mg/kg, potentiated SW-SIA dose-dependently and did not affect FS-SIA at the doses employed. Both bicuculline, a GABAA receptor antagonist, 0.5 to 2.0 mg/kg, and picrotoxin, a Cl- channel blocker, 0.25 to 1.0 mg/kg, dose-dependently suppressed PSY- and FS-SIA. Meanwhile, the effects of both drugs on SW-SIA were less than those on PSY- and FS-SIA, namely, bicuculline slightly inhibited it only at 2.0 mg/kg, and picrotoxin did not produce any appreciable effect even at the highest dose. Baclofen, a GABAB receptor agonist, at 5.0 and 10.0 mg/kg had no influence on each SIA. On the contrary, CGP 35348, a GABAB receptor antagonist at 20 to 100 mg/kg caused the dose-dependent blockade of FS-SIA, but affected neither PSY- nor SW-SIA. The production of PSY- and SW-SIA is attributable to the GABAA receptors/Cl- channel mediated mechanism alone, while that of FS-SIA involves both GABAA and GABAB receptor mediated systems. Thus, GABAergic systems play an important role in the production of each SIA; however, the participation of the receptor subtypes in the mechanism was different from each other.     

The GABAA-BZR complex as target for the development of anxiolytic drugs

Anxiety disorders have been linked to alterations in γ-aminobutyric acid (GABA) neurotransmission. GABA interacts with the ligand-gated ion channels, GABAA receptor (GABAA-R) subtypes, and regulates the flow of chloride into the cell, causing neuron hyperpolarization. GABAA-Rs are assembled from a family of 19 homologous subunit gene products and form mostly hetero-oligomeric pentamers. The major isoforms of the GABAA-Rs contain α, β and γ subunits and show a regional heterogeneity that is associated with distinct physiological effects. A variety of allosteric ligands can modulate the response to GABA by binding at different sites on the GABAA-R complex. The best characterized binding site is the benzodiazepine (BZ) one, which is located at the α/γ subunit interface. BZs are commonly used in therapy for their effects as anxiolytic, anticonvulsants, myorelaxants and hypnotics. The broad range of pharmacological effects of classical BZs are mediated by the selective activation of different GABAA-R subtypes: the α1 subunit containing BZ receptor (BZ-R) mediates sedation, the α2 and α3 subunit containing BZ-R mediates anxiolysis and myorelaxation, and the α5 subunit containing BZ-R mediates cognitive impairment. Based on the current understanding of the diversity of the GABAA-R family, different approaches have been employed to develop drugs that target the GABAA/BZ-R complex with selective anxiolytic action and improved profiles. In this review, we present current knowledge about the role of the GABAA/BZ-R complex in anxiety disorders, new insights into the molecular biology of the receptor complex, and the importance of this target in the development of new therapeutic agents in anxiety.     

An N-terminal histidine is the primary determinant of alpha subunit-dependent Cu2+ sensitivity of alphabeta3gamma2L GABA(A) receptors

Copper (Cu2+) is a physiologically important cation and is released from nerve terminals. Cu2+ modulates GABAA receptor currents in an alpha subunit subtype-dependent manner; alpha1beta3gamma2L receptors are more sensitive to Cu2+ than alpha6beta3gamma2L receptors. We compared the effect of Cu2+ on alphabeta3gamma2L receptors containing each of the six alpha subtypes and generated alpha1/alpha6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for alpha subtype-dependent differences in Cu2+ sensitivity. Whole-cell GABAA receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant alpha subunits with beta3 and gamma2L subunits. Maximal Cu2+ inhibition of alpha1beta3gamma2L and alpha2beta3gamma2L receptor currents was larger (52.2 +/- 3.0 and 59.0 +/- 2.5%, respectively) than maximal inhibition of alpha3beta3gamma2L, alpha4beta3gamma2L, alpha5beta3gamma2L, and alpha6beta3gamma2L receptor currents (22.6 +/- 3.1, 19.2 +/- 3.4, 20.2 +/- 4.8, and 21.2 +/- 3.6%, respectively). Receptors containing chimeric constructs with alpha1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu2+ to an extent similar to those with alpha1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu2+ sensitivity. alpha1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the alpha2 subtype) conferred higher Cu2+ sensitivity, and the H141 residue was the major determinant in the motif. The beta3 subtype M2 domain residue H267, which is a major determinant of Zn2+ inhibition, and alpha6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn2+ sensitivity of the alpha6 subtype, also seemed to contribute to Cu2+ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in alpha1 and alpha2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu2+, that residue H141 is the major determinant in that motif, and that Cu2+ may also interact with GABAA receptors at sites similar to or overlapping Zn2+ sites.     

Cultured Hippocampal Pyramidal Neurons Express Two Kinds of GABAA Receptors

We combined a study of the subcellular distribution of the alpha1, alpha2, alpha4, beta1, beta2/3, gamma2, and delta subunits of the GABAA receptor with an electrophysiological analysis of GABAA receptor currents determine the to types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that alpha1, alpha2, beta2/3, and gamma2 subunits formed distinct clusters of various sizes, which were colocalized with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22 to 58%. In contrast, alpha4, beta1, and delta subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA receptor currents were moderately sensitive to GABAA and were modulated by diazepam. The whole-cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn2+, furosemide, and loreclezole; they were insensitive to diazepam. These studies suggest that two kinds of GABAA receptors are expressed on cultured hippocampal neurons. One kind of receptor formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The alpha1, alpha2, beta2/3, and gamma2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The alpha4, beta1, and delta subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing alpha4, beta1, and delta subunits.     

NMDA receptor pharmacology: perspectives from molecular biology

The NMDA receptor is an important target for drug development, with agents from many different classes acting on this receptor. While the severe side effects associated with complete NMDA receptor blockade have limited clinical usefulness of most antagonists, the understanding of the multiple forms of NMDA receptors provides an opportunity for development of subtype specific agents with potentially fewer side effects. Different NMDA receptor subtypes are assembled from combinations of NR1 and NR2 subunits with each subunit conveying distinct properties. The NRI subunit is the glycine binding subunit and exists as 8 splice variants of a single gene. The glutamate binding subunit is the NR2 subunit, which is generated as the product of four distinct genes, and provides most of the structural basis for heterogeneity in NMDA receptors. Pharmacological heterogeneity results from differences in the structure of ligand binding regions, as well as structural differences between subtypes in a modulatory region called the LIVBP-like domain. This region in NR1 and NR2B controls the action of NR2B-selective drugs like ifenprodil, while this domain in receptors containing the NR2A subunit controls the action of NR2A-selective drugs such as zinc. This suggests that NMDA receptor subtype selective drugs can be created, and further understanding of subtype specific mechanisms ultimately may allow successful use of NMDA receptor antagonists as therapeutic agents.     

Honokiol and magnolol selectively interact with GABAA receptor subtypes in vitro.

Honokiol and magnolol have been identified as modulators of the GABAA receptors in vitro. Our previous study suggested a possible selectivity of honokiol and magnolol on GABAA receptor subtypes. This possibility was examined in the current study by 3H-muscimol and 3H-flunitrazepam binding assays on various rat brain membrane preparations and human recombinant GABA(A) receptor subunit combinations expressed by the Sf-9/baculovirus system. Generally, honokiol and magnolol have a similar enhancing effect on (3)H-muscimol binding to various membrane preparations in nonsaturation binding assays. Honokiol and magnolol preferentially increased (3)H-muscimol binding to hippocampus compared to cortex and cerebellum (with a maximum enhancement of 400% of control). As for subunit combinations, honokiol and magnolol have a more potent enhancing effect on alpha2 subunit containing combinations (with a maximum enhancement of 400-450% of control). This action was independent of the gamma subunit. In saturation binding assays, magnolol affected either the number of binding sites (ca. 4-fold on alpha2 containing combinations) or the binding affinity (on alpha1 containing combinations) of (3)H-muscimol binding to various GABAA receptor subunit combinations. In contrast, honokiol increased only binding sites on alpha2beta3gamma2s and alpha2beta3 combinations, but both the number of binding sites and the binding affinity on alpha1beta2gamma2S and alpha(1)beta2 combinations. These results indicate that honokiol and magnolol have some selectivity on different GABAA receptor subtypes. The property of interacting with GABAA receptors and their selectivity could be responsible for the reported in vivo effects of these two compounds.     

Techniques: Use of concatenated subunits for the study of ligand-gated ion channels

Members of the pentameric ligand-gated ion channel family, comprising GABA(A) receptors, nicotinic acetylcholine receptors, glycine receptors and 5-HT3 receptors, are involved in information transfer at both synapses and the neuromuscular junction. However, receptors that are composed of five subunits are difficult to analyse by recombinant expression of a mixture of the single subunits because multiple receptor subtypes with different subunit composition or arrangement can be formed. Covalently linking the C-terminus of the preceding subunit with the N-terminus of the following subunit to form a concatenated subunit enables the precise predetermination of subunit arrangement in these receptors. A forced subunit assembly enables the characterization of: (i) receptor architecture; (ii) properties of receptors that contain different subunit isoforms in specific locations; and (iii) selective introduction of a mutation into a specific subunit that occurs multiple times in a receptor. Thus, this method also facilitates the investigation of positional effects of mutations associated with diseases.     

The effects of subunit composition on the inhibition of nicotinic receptors by the amphipathic blocker 2,2,6,6-tetramethylpiperidin-4-yl heptanoate

The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs that may be selective for their ability to activate or inhibit a limited range of nicotine acetylcholine receptor subtypes. In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that is a potent inhibitor of neuronal nicotinic receptors. Evaluation of nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus laevis oocytes indicated that TMPH can produce a potent and long-lasting inhibition of neuronal nAChR formed by the pairwise combination of the most abundant neuronal alpha (i.e., alpha3 and alpha4) and beta subunits (beta2 and beta4), with relatively little effect, because of rapid reversibility of inhibition, on muscle-type (alpha1beta1gammadelta) or alpha7 receptors. However, the inhibition of neuronal beta subunit-containing receptors was also decreased if any of the nonessential subunits alpha5, alpha6, or beta3 were coexpressed. This decrease in inhibition is shown to be associated with a single amino acid present in the second transmembrane domain of these subunits. Our data indicate great potential utility for TMPH to help relate the diverse central nervous system effects to specific nAChR subtypes.