Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X

GABAA receptors as novel drug targets for treatment of mental disorders

Abolghasem Esmaeili1,*, Kamran Ghaedi1

1Cell, Molecular & Developmental Biology Division, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

*Corresponding Author: email address: [email protected] (A. Esmaeili)

ABSTRACT

A balance between excitatory and inhibitory neurotransmissions in brain is an essential factor for the proper function of the brain. The amino acid gamma-aminobutyric-acid (GABA) is considered as the major inhibitory neurotransmitter in brain. Thus, GABAergic neurons play a key role in regulating behavior. Previous data have revealed the complex subunit structural design for GABAA receptor channel, in which a pentameric assembly resulting from 5 of at least 21 subunits, grouped in the eight classes alpha (α1-6), beta (β1-4), gamma (γ1-4), delta, pi (π), epsilon (ε), theta (θ) and rho (ρ1-3) permits an immense number of putative receptor isoforms. GABAARs are highly diversed in the central nervous system in which this diversity may be related to some mental disorders. Any alteration in expression of the GABAA receptor causes neurophysiological and functional consequences that might be associated with neurological disorders. Some neuropsychiatric disorders, such as anxiety, epilepsy and sleep disorders, are effectively treated with therapeutic agents that act on the GABAA receptor. In this article, the contribution of GABAA receptor deficits to central nervous system disorders, in particular anxiety disorders, epilepsy, schizophrenia and insomnia, will be reviewed. The better understanding of GABA and its receptors may help us to find novel therapeutic agents for treatment of mental disorder in future research.

Keywords: Epilepsy; Anxiety; Insomnia; Schizophrenia; GABAA receptor subtypes

INTRODUCTION functional channels while, gamma subunits are The amino acid gamma-aminobutyric-acid required to mimic the full repertoire of native (GABA) is the major inhibitory receptor for responses to drugs. The neurotransmitter in the CNS [1, 2] that knowledge of the complex pharmacology of mediates most of its effects through receptors GABAA receptors might eventually enable termed GABAA. Previous data have revealed site-directed drug design to elaborate our the complex subunits structural design of this understanding of GABA-related disorders and receptor channel, in which a pentameric of the complex interaction of excitatory and assembly resulting from five of at least 21 inhibitory mechanisms in neuronal processing. subunits, grouped in the eight classes alpha To understand the role of GABAA receptors in (α1-6), beta (β1-4), gamma (γ1-4), delta, pi, mental disorder many methods including epsilon, theta, and rho (ρ1-3) [3-5] permits an molecular and electrophysiological techniques immense number of putative receptor have been used. In the present study we have isoforms. These varieties are extended the shown the role of GABAARs in some mental existence of several splicing variant forms, for disorders. instance of the α6, β2 and γ2 subunits [6]. The subunit combination of GABAA receptor Structure and molecular biology of GABA determines the specific effects of allosteric receptors modulators as (BZs), The ligand-gated ion channels (LGICs) , steroids, and general anaesthetics, super family share a common proposed some convulsants, polyvalent cations, and structure. They have a long extracellular ethanol. These agents act through different amino terminus (around 200 amino acid), binding sites some of which are not identified thought to be responsible for ligand channel yet [7, 8]. Drugs and endogenous ligands bind interactions and forms agonist binding site, either to the extracellular domain or channel and four transmembrane (TM) domains and a domain of the GABAA receptors and act as large intracellular domain between TM3 and positive or negative allosteric modulators [9]. TM4 [2, 10]. The TM3-TM4 loop is an In heterologous expression systems, the important site for regulation by presence of alpha, beta subunits are needed for phosphorylation and for localization at

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Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X synapses [11]. It is thought that TM2 forms the and 4 genes, which are separated by less than lining segment of the . The 60-kb, are arranged in the head to head extracellular amino terminus is believed to orientation [17]. The 1 transcription unit faces incorporate neurotransmitter and some the tail of 2. The , and 1 subunit modulator binding sites and it contains a mRNA are predominately expressed in the conserved motif, the so-called Cys loop undifferentiated neuroepithelium of rat embryo (cysteine loop). The Cys-loop is characterized [18]. During postnatal development, down by two cysteine residues spaced by thirteen regulation of the , and 1 subunits otherwise largely divergent amino acid occurs in some specific region whereas residues (Figure.1). This structure is common expression levels of most other subunits within acetylcholine (ACh), glycine and type 3 increase. Highly expression of the , and serotonin (5-HT3) receptor as well. 1 genes in the hippocampal formation of the

adult rat [19], shows that cluster organization may be necessary to preserve region-specific transcription. The most abundant GABAA receptor subunit genes encoding are found in a 2-  cluster on 5q31.2-q35 [17, 20-24]. The 2 and 6 genes are separated by less than 60-kb, with transcription units facing in opposite directions [17]. The colocalization of the , and 2 subunits may be related to their coordinate gene regulation throughout the nervous system; however, direct observation of Figure 1. Ligand gated ion channels are composed of five peptide units. Each unit has an N-terminal domain, four coordinate regulation has not been reported. transmembrane domains, a large extracellular loop The 6 gene that is head-to-head with 2 is between M3 and M4 and a C-terminus [116]. expressed only in cerebellar granule cells [25], suggests that there is also independent Channels in GABAA and glycine receptors are regulation of transcription for individual anion-selective, whereas in the ACh and 5- members in this gene cluster. HT3 receptors are cation-selective [12]. There The  GABAA receptor subunit is a significant in each genes are localized on chromosome 15q11– receptor gene family. In GABA receptor A q13 [17, 26-31]. Lastly, the putative  subunits the region between TM3 and TM4 gene cluster on chromosome Xq28 is shows a little or no sequence homology, analogous to the cluster on chromosome 15 suggesting that this domain can tolerate many [16, 17, 32, 33]. The -subunit gene is changes without affecting any possible analogous to , and  has the same position functional role [13, 14]. GABAA receptors are large (450-627 amino acids in length) and transcriptional orientation as the -subunit embedded in the cell membrane of neurons. gene in other clusters. The channel is formed in the centre of receptor Gene expression can be controlled at that consists of five molecules, or multiple levels of transcription, alternative splicing, mRNA stability, translation, post- subunits [15]. GABAA receptors are activated directly by GABA. In this phenomenon they translational modification, intracellular mediate fast response to GABA and open trafficking, and protein degradation. However, channel to allow the inward passage of gene regulation is predominantly controlled at chloride and bicarbonate ions from outside the the level of transcription initiation [34, 35]. cell to inside it. Any abnormal alteration during gene expression could result in changes in function Chromosomal localization of GABAA and could lead to mental disorders. receptor genes It has been found that each GABAA Distribution of GABAA receptor subunits in receptor subunit is encoded by homologous, the CNS distinct genes. Many of the subunit genes are A variety of studies using in situ organized in  and  gene hybridization [25, 36] and clusters on different [16, 17]. In immunohistochemistry [37-41] have indicated humans, the  subunit genes are the distribution of GABAA receptor subunits in localized on chromosome 4p14-q12. The 1 the brain. The GABAA receptor α1, β1, β2, β3,

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Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X and γ2 are found throughout the brain with 23% [43]. These results are in a good different distribution levels. The α2, α3, α4, agreement with in situ hybridization data α5, α6, γ1, and δ subunits are found in certain which showed in the brain that γ1 and γ3 regions of the brain (Table 1). Mehta et al mRNA are expressed in much smaller amounts (1999) reported the percentage of the binding relative to γ2 mRNA [25, 36, 44]. Although sites immunoprecipitated by antisera to these data do not show the regional various subunits of GABAA receptors in the distribution of GABAA receptor subunits, they adult brain regions. These percentages are as show which subunits are abundant in the brain. follows: α1 = 70-90%, α2 = 4-28%, α3 = 12- Distribution of GABAA receptor subunits in 24%, α4 = 0-15%, α5 = 4-14%, α6 = 30-39% the brain are summarised in table 1. Gaba (cerebellum), β1 = 2-32%, β2 = 55-96%, β3 = receptor also could be seen in other tissue far 19-52%, γ1 = 0-19%, γ2 = 50-94%, γ2S = 31- from CNS such as sperm [45]. 52%, γ2L = 37-65%, γ3 = 0-18%, and δ = 0-

Table 1. Regional distributions of GABAA receptor subutints in the brain (Sieghart, 2002)

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Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X

Pharmacology of GABAA receptors targets for many important drugs such as More than one hundred agents act on benzodiazepines, general anaesthetics, GABAA receptors. These agents act in steroids, convulsants and barbiturates [51]. different binding sites which some are not Benzodiazepines mediate their sedative, known yet [46]. Drugs and endogenous amnesic and anxiolytic actions via binding ligands act as positive or negative to GABAA receptors. The sensitivity of allosteric modulators on GABAA GABA receptors to benzodiazepines receptors. Compounds bind on either depends on the GABA receptor subunits extracellular domain or channel domain of composition and is different from one the receptor and act as positive or negative region of the brain to another [52]. It is modulators [2]. Benzodiazepines are suggested that  subunit has a role as positive modulators acting on the important as subunit in determining extracellular domain, whereas -carbolines benzodiazepines sensitivity [53]. Different act mainly as negative modulators. The variants of the  subunit can influence both presence or absence of the 2 subunit in affinity and efficacy of various the structure of the GABAA receptor can benzodiazepines site ligands and also the influence the action of positive allosteric type of  subunit can influence the affinity modulators [47]. The effect of compounds and efficacy of a number of bind on channel the domain can be benzodiazepines such as . positive or negative. Barbiturates and Receptor combinations which include the steroid hormones act as positive allosteric 1 receptor subunits display, modulators, whereas pregnenolone and benzodiazepines type one pharmacology act as negative modulators. and bind , zolpidem and other However their effects do not depends on benzodiazepines with high affinity, while the structure of the receptors [47-49]. those receptors including the 2, 3, and Benzodiazepines have been widely 5 subunits display benzodiazepines type prescribed since chlordiazepoxide was first two pharmacology and confer low affinity introduced in 1960; and because of their for this ligand, whereas the 4 and 6 safety and efficacy benzodiazepines subunits confer benzodiazepines became the most prescribed drugs in the insensitivity [54]. The crucial amino acids 1960s and 1970s. However, because of responsible for potent action of benzodiazepines side effects and drug benzodiazepines are located within the N- abuse the use of benzodiazepines have terminal domains of  and , within the fallen in recent years, but they are still putative second and third transmembrane highly prescribed drugs [50]. domains [55]. In one study various Benzodiazepines the one of these agents compounds of benzodiazepines were use to treat anxiety have side effects such examined on three different human as sedation, ataxia, amnesia, tolerance, and GABA receptor combinations (21, physical dependence. Because the A physiological and pharmacological role of 212S, 211). The data showed significant differences among these various native GABAA receptor assemblies is not yet known, it is not easy to GABAA combinations. The 1 subunit in synthesize compounds selective for comparison to 2 subunit can confer a particular receptor assembly to get a different pharmacological profile with desired therapeutic effect without any regard to site ligands [53]. serious side effect. All GABA receptors The patch-clamp technique will be used to are sensitive to GABA. The binding of facilitate the further characterisation of GABA to specific sites on the receptor these drugs and their GABAA receptors. In results in the opening of an intrinsic ion this thesis the effects of some of the drugs channel and the flux of chloride into the on GABAA channels was examined. cell, which leads to a hyperpolarization of Understanding the basic mechanism of the cell membrane and an increase in the operation of the GABAA receptors may possibly result in more specific drugs and inhibitory tone. The GABAA receptors are

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Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X better treatments of some of the mental whereas negative modulators create disorders. One way make pharmacological anxiogenic-like effects [68]. Various GABA screening of GABA subunits easy is using analogs and agents affecting transmitter an anion-sensitive yellow fluorescent metabolism to enhance GABAergic tone have protein-based assay [56] also been reported to exert anxiolytic effects [71, 72]. Chronic alcoholism leads to localized The action of nM and M concentration brain damage, which is eminent in superior of diazepam on 2 recombinant frontal cortex but mild in motor cortex. The expressed into Xenopus oocytes is probability of developing dependence different at low concentration of GABA is associated with genetic markers. GABAA [55]. At M concentration diazepam action receptor expression differs between alcoholics is independent of the 2 subunit. Using and controls [73, 74]. mutation of rho subunit of corresponding Many researchers guess that GABAergic TM2 and TM3 residues and application of dysfunction plays an important role in the mechanism of neural impairment in Angelman diazepam support this results. Together syndrome [75, 76]. these data suggest that diazepam, at low First round reports have showed altered concentration of GABA creates two expression of GABA receptors in the brains of distinct components of potentiation. subjects with autism suggesting Gamma subunits are relatively insensitive GABA/glutamate system dysregulation. to Zn2+ compared to subunit receptors Significant decreases in GABRA1, GABRA2, [57]. GABRA3, and GABRB3 in parietal cortex have been reported. These results reveal that GABAA receptors and mental disorder GABAA receptors are reduced in the brain Studies on GABAA receptors show that regions that have previously been associated in these receptors are involved in the pathology the pathogenesis of autism, suggesting of several neurological and psychiatric widespread GABAergic dysfunction in the diseases, such as epilepsy, anxiety, alcoholism brains of subjects with autism [77, 78]. [58], Angelman’s syndrome [59], autism [60], Adult neurogenesis adjusts plasticity and depression [61], premenstrual syndrome [62- function in the hippocampus, which is critical 64], sleep disorders [65], and Alzheimer’s for memory and vulnerable to Alzheimer's disease [64]. Some psychiatric disease such as, disease (AD). Promoting neurogenesis may spasticity, and stiff-person syndrome, are improve hippocampal function in AD brains. related to lack of GABAergic function in the However, how amyloid β (Aβ), the key AD brain. pathogen, affects the development and GABA transmission plays a key role in function of adult-born neurons remains controlling seizure activity. The exact nature unknown. Adult-born granule cells (GCs) in of its effect depends on the particular position human amyloid precursor protein (hAPP) in the brain and the pathway involved. Animal transgenic mice, an AD model, showed greater studies have helped to describe specific brain dendritic length, spine density, and functional regions such as the substantia nigra that are responses than did controls early in vital in controlling seizure activity. development, but were impaired Antiepileptic drugs such as vigabatrin, a drug morphologically and functionally during later developed to treat resistant epilepsy, can maturation. Early inhibition of GABAA increase GABA transmission in these regions receptors to suppress GABAergic signaling or and may thereby afford seizure protection late inhibition of calcineurin to enhance [66]. glutamatergic signaling normalized the The role of gamma-aminobutyric acid development of adult-born GCs in hAPP mice (GABA) in depression and anxiety has been with high Aβ levels. Aβ-induced increases in described. New data from both animal and GABAergic neurotransmission or an human experimentation have helped define the imbalance between GABAergic and key role for this transmitter in both these glutamatergic neurotransmission may mental pathologies [67]. Dysfunction of the contribute to impaired neurogenesis in AD gamma-aminobutyric (GABA) in central [79]. nervous system has long been associated with In comparison to glutamatergic and anxiety disorders [68-70]. In both human and cholinergic systems, the GABAergic system is animal studies, positive modulators of GABA relatively spared in AD, but the precise receptors generally possess anxiolytic activity, mechanisms underlying differential

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Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X vulnerability are not well understood. Using temporal lobe epilepsy (TLE), alterations in several methods, investigations demonstrate GABA receptor binding have been that despite resistance of the GABAergic documented [98]. In the CA2 area of TLE system to neurodegeneration, particular patients there is a uniform increase in 1, 2, subunits of the GABAA receptor are altered 2/3, and 2 subunits in the dentate granule with age and AD, which can induce cell layer, while levels of 2 alone increase. compensatory increases in GABAA receptor Moreover, extensive cell loss in the CA1 and subunits within surrounding cells. Although CA3 regions is accompanied by a significant alteration in GABAA may be diffident and decrease in the  subunit. Mutations in the perhaps low, this may be enough for alteration GABAA receptor 2 subunit [99, 100] and 1 in the pharmacokinetic and physiological subunit [101, 102] have been described in properties of the receptor. Therefore, it is patients with epilepsy. critical to understand the subunit composition Innate genetic errors caused by dysfunction of of individual GABAA receptors in the diseased the GABAergic system have features common brain when developing therapeutics that act at in many mental disorders. Patients with 1p36 these receptors [80]. deletions, missing a series of genes including

the delta subunit of the GABAA receptor, show GABAA receptors alteration in CNS neurological and neuropsychiatric anomalies Genetics variations in gene expression may [103]. The genes encoding the alpha 5, beta 3 be associated with mental disorders. An and gamma 3 subunits of the GABAA receptor altered expression of the GABAA receptor has on chromosome 15 are commonly deleted in neurophysiologic and functional consequences patients with Prader-Willi or Angelman that might relate to the behavioral and syndrome [104] that this altered epileptic phenotype associated with fragile X neurobehavioral function of Prader-Willi syndrome, such as anxiety, depression, patients could arise directly from an altered epilepsy, insomnia, and learning and memory GABAA receptor composition and expression. [81, 82]. Alteration in GABAergic inhibitory In addition, beta 3 mutant mice display a action such as alterations in the number of phenotype similar to some aspects of GABAA receptors [4], alterations in GABAA Angelman syndrome, including epilepsy, receptor subunit composition [40, 83, 84], hyperactivity, learning and memory deficits, increased sensitivity to Zn2+ inhibition of and poor motor skills [105]. It is suggested GABAA receptors in the dentate granule cells that the associated seizures in some [84-86], decreases in GABA transporter experimental mutant mice could be elucidated function [87] disconnection of inhibitory by an imbalanced neurotransmitter interneurons from excitatory inputs [88] and concentration that led to a pathological use-dependent reduction of excitatory drive to adjustment of the GABA inhibitory system inhibitory interneurons [89] is important in the [106, 107]. Significant decrease in mRNA generation of epilepsy. Alterations in GABAA expression of GAD67, delta alpha 1, alpha 3 receptor subunit gene expression also may be and alpha 4, beta 1 and beta 2, and gamma 1 important in mediating Alzheimer’s disease and gamma 2 subunits of the GABAA receptor, [90, 91], schizophrenia [92-95], and ischemia has been demonstrated in the fragile X mouse [96, 97]. In severe cases of Alzheimer’s [82, 108, 109]. disease, levels of 1 subunit protein are significantly reduced compared with mild Agents effect on GABAA receptors in cases within the hippocampal subregions CA1, mental disorder CA2, and prosubiculum, but not in the dentate Many drugs that work on the GABAA gyrus, subiculum, and presubiculum [91]. receptor are commercially available. GABAA Furthermore, level of 3, but not 2, mRNA is receptors are targets of both various classes of reduced in the pathologically severe group in clinically relevant drugs, including all hippocampal subregions except CA4 [90]. benzodiazepines, barbiturates, and general GABAA receptor subunit mRNA are anaesthetics, in addition to endogenous differentially regulated in the prefrontal cortex components, such as neuroactive steroids, all of schizophrenics. While modest reductions in of which allosterically modulate receptor , and 2 subunit mRNA function [5, 110]. have been detected by in situ hybridisation Benzodiazepines are widely used as anxiolytic histochemistry [93], reduced levels of 2S agents in many countries even though transcripts have been observed using antidepressants are now suggested as the first semiquantative RT-PCR [94]. In human

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Journal of Paramedical Sciences (JPS) Summer 2010 Vol.1, No.3 ISSN 2008-496X choice of treatment for anxiety [111], largely mammalian central nervous system. J Child due to their safety profile. Benzodiazepines are Neurol 2003;18(1):39-48. very effective in short-term use. A point 2. Olsen RW, Tobin AJ. Molecular biology of mutation in the GABAA receptor 2 subunit GABAA receptors. Faseb J 1990; 4(5):1469- in transgenic mouse lines selectively disrupted 80. the anxiolytic effects of benzodiazepines, but 3. Darlison MG, Pahal I, Thode C. not other pharmacological effects of Consequences of the evolution of the benzodiazepines [112]. This demonstrates a GABA(A) receptor gene family. Cell Mol key role of the GABAA receptor 2 subunit in Neurobiol 2005; 25(3-4):607-24. anxiolytic effects of benzodiazepines. 4. Whiting PJ. The GABA-A receptor gene Some neuropsychiatric disorders, such as family: new targets for therapeutic anxiety, epilepsy, sleep disorders and intervention. Neurochem Int 1999; 34(5):387- convulsive disorders, have been effectively 90. treated with therapeutic agents that enhance 5. Barnard EA, Skolnick P, Olsen RW, Mohler the action of GABA at the GABAA receptor in H, Sieghart W, Biggio G, et al. International nervous tissue [113]. Union of Pharmacology. XV. Subtypes of Expression of specific subtypes of the gamma-aminobutyric acidA receptors: GABAA receptor decrease in fragile X classification on the basis of subunit structure syndrome; it is indicate that specific enhancers and receptor function. Pharmacol Rev 1998; of this receptor might be suitable drugs to treat 50(2):291-313. the behavioral aspects of the disorder. These 6. Korpi ER, Grunder G, Luddens H. Drug compounds with partial agonist properties interactions at GABA(A) receptors. Prog were already reported to have improved Neurobiol 2002; 67(2):113-59. profiles in whole animal behavioral models 7. Johnston GAR. GABA(C) receptors: [114]. Relatively simple transmitter-gated ion The GABA transporter 1 is another novel channels? Trends in Pharmacological Sciences candidate as a promising target for treatment 1996; 17(9):319-23. of anxiety disorders with panic symptoms 8. Johnston GAR. GABA(A) receptor [115]. pharmacology. Pharmacology & Therapeutics 1996; 69(3):173-98. CONCLUSION 9. Olsen RW, Tobin AJ. Molecular-Biology of GABA system as a whole and especially Gaba-a Receptors. Faseb Journal 1990; GABAA receptors have important roles in CNS 4(5):1469-80. and have also been strongly linked to several 10. Schofield PR, Darlison MG, Fujita N, Burt mental disorders such as epilepsy, anxiety, DR, Stephenson FA, Rodriguez H, et al. depression and alcoholism with different Sequence and functional expression of the mechanisms. Previous research found GABA A receptor shows a ligand-gated evidences for association of various GABA receptor super-family. Nature 1987; receptor genes and a range of mental disorder- 328(6127):221-7. related phenotypes. Alteration in GABAergic 11. Brandon N, Jovanovic J, Moss S. Multiple inhibitory action such as alterations in the roles of protein kinases in the modulation of number of GABAA receptors, alterations in gamma-aminobutyric acid(A) receptor function and cell surface expression. GABAA receptor subunit composition and gene expression of the GABAA receptor has Pharmacol Ther 2002; 94(1-2):113-22. neurophysiologic and functional consequences 12. Akabas MH. GABAA receptor structure- that might relate to mental disorders. function studies: a reexamination in light of Therefore, the better understanding of new structures. Int Rev Neurobiol 2004; 62:1-43. relationship of GABAA receptors and mental disorders potentially could help to find novel 13. Whiting PJ, Bonnert TP, McKernan RM, drugs to overcome aforementioned disorders Farrar S, Le Bourdelles B, Heavens RP, et al. and avoid their side effects. Molecular and functional diversity of the expanding GABA-A receptor gene family. REFERENCES Ann N Y Acad Sci 1999; 868:645-53. 1. Simeone TA, Donevan SD, Rho JM. 14. Moss SJ, Smart TG. Constructing Molecular biology and ontogeny of gamma- inhibitory synapses. Nat Rev Neurosci 2001; aminobutyric acid (GABA) receptors in the 2(4):240-50.

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