GABA (γ-Aminobutyric Acid)
GABA Synthesis and Metabolism
As a hydrophilic molecule, GABA, in general, cannot cross the blood brain barrier. It is produced within the brain from l-glutamate by a metabolic pathway termed GABA shunt whose aim is to both produce and conserve the supply of GABA. GABA is also synthesized in other, non-neuronal tissues (e.g., immune cells, retina, pancreas, lung, etc.). The main precursor for GABA production in vivo is glucose, although some amino acids and pyruvate may likewise serve that role. The first step in the production of GABA is the transamination of α-ketoglutarate (formed in the Krebs cycle from glucose) by GABA α-oxoglutarate transaminase (GABA-T) into l-glutamic acid. The second step involves glutamic acid decarboxylase (GAD), which catalyzes the decarboxylation of l-glutamic acid to GABA by using pyridoxal phosphate as a cofactor (Roth et al. 2003). GABA is metabolized by GABA-T to succinic semialdehyde. This transamination process is tightly regulated: to assure GABA availability, reaction occurs when α-ketoglutarate, the parrent compound is present and thus able to accept the amino group from GABA, reforming again glutamic acid. Hence, to conserve the available supply of GABA, for each molecule of GABA metabolized, a molecule of precursor is reformed (Olsen and DeLorey 1999).
Regulation of GABA Synthesis by GAD Isoforms
In mammals, there are two isoforms of GAD whose expression is differentially regulated during development. Isoforms have distinct subcellular localization, regulatory mechanisms, patterns of interaction with cofactors, and actions in the brain (Kaufman et al. 1991). Isoform GAD67 is expressed early during development, while the expression of GAD65 starts later. This pattern of expression is in accordance with their relative roles in synaptogenesis and neurotransmitter synthesis. GAD67 is constitutively active and is responsible for the basal GABA production, while GAD65 is transiently activated in response to demands for GABA in neurotransmission (Somogyi et al. 1995; Wei and Wu 2008). GAD65 is associated with synaptic vesicle membranes in axonal nerve endings and synthesizes GABA for neurotransmission. Hence, this isoform mediates fast, point-to-point GABAergic neurotransmission, while GAD67 is evenly distributed throughout the neuronal cytoplasm and synthesizes GABA which is used as a trophic factor for synaptogenesis during neuronal development, as a neuroprotective agent, for the regulation of reactive molecules during oxidative stress, or as an energy source via GABA-dependent metabolic shunt in brain mitochondria through which 10–20% of glucose metabolism may flow. This shunt allows four out of five carbons in the form of 2-oxoglutarate to re-enter Krebs cycle at the level of succinate (Schousboe et al. 1997).
GAD is regulated by transcriptional/translational and posttranslational mechanisms. The adjustment of GAD expression at the transcriptional level and the regulation of posttranslational modifications are the key mechanisms in the control of GABAergic neurotransmission. Possibilities for posttranslational regulation of GAD include protein phosphorylation, palmitoylation, and activity-dependent cleavage (Modi et al. 2015). Distinct variations of GABA or GAD levels have been connected to different neurological disorders, including anxiety, schizophrenia, and epilepsy as well as Huntington’s and Parkinson’s disease. GAD65 has been considered as an autoantigen in certain autoimmune diseases in humans such as insulin-dependent diabetes, mellitus, Stiff-Person syndrome, and Batten disease.
Site of GABA Action
GABA is present in high concentrations (millimolar) in many brain regions highlihgting the specific and demanding actions of GABAergic neurons. Local-circuit interneurons that constitute 15–20% of all cortical neurons use GABA as the neurotransmitter (Löscher and Frey 1982). GABAergic neurotransmission at the presynaptic sites is tuned by the rate of GABA synthesis, vesicular release, and reuptake mechanisms, while the interactions of GABA with its receptors are limiting factor in GABA transmission at the postsynaptic sites. GABA interacts with two types of receptors to achieve effects: ionotropic GABAA receptors (fast-acting ligand-gated chloride channels) and metabotropic GABAB receptors (indirectly coupled via G proteins to calcium or potassium channels). These receptors can be localized pre- or postsynaptically. GABAergic transmission is terminated by reuptake of GABA via GABA transporters (GATs) in presynaptic nerve terminals and at a lesser extent into surrounding glial cells, mainly astrocytes. The function of GATs is dependent on the extracellular availability of sodium ions with an additional dependence on chloride ions. GATs can transport GABA in two directions: into the cells, or into the extracellular fluid. GABA transported into nerve terminals can be reutilized and mainly constitutes the vesicular GABA pool, whereas only a smaller fraction enters metabolic pathway. GABA absorbed back into nervous terminals in some cases can be reused by circuitous pathways, including the Krebs cycle. GABA transported into astrocytes is metabolized to succinic semialdehyde by GABA-T and cannot be resynthesized since glial cells lack GAD. GABA in astrocytes can be converted to glutamine and further by glutaminase activity to glutamate that reenters GABA synthetic pathway (McGeer and McGeer 1989).
GABA acts via two major classes of receptors that differ in terms of their pharmacological, electrophysiological, and biochemical characteristics. Most of its physiological effects GABA exerts through binding to GABAA receptors, which are mainly embedded in postsynaptic membrane and mediate transient, fast synaptic inhibition that occurs in milliseconds. Electrophysiological studies revealed that GABAA receptors mediate GABA action by enhancing membrane conductance, followed by a membrane hyperpolarization. This reduces probability that the postsynaptic cell will generate an action potential and results in neuronal inhibition. In particular, GABA released into the synaptic cleft from the presynaptic terminals diffuses and binds to postsynaptically located GABAA receptors, which in turn undergo rapid conformational changes. GABA binding causes opening of the integral chloride ion channel of the receptor and allows chloride ions to flow down their gradient across the postsynaptic membrane. In contrast to postsynaptically placed GABAA receptors, extrasynaptically located receptors are sensitive to very low, ambient concentrations of GABA and mediate long-term inhibition (Knoflach et al. 2016). GABAA receptors are hetero-oligomeric pentamers assembled from the pool of 19 homologous subunits. Their pharmacological characteristics depend on subunit composition, topography, localization, and specific role of brain circuits (Whiting et al. 1999). A variety of clinically important drugs, including anticonvulsants, anxiolytics (e.g., benzodiazepines), general anesthetics, barbiturates, ethanol, and neuroactive steroids, as well as drugs important as research tool such as convulsive picrotoxin, allosterically modulating GABA-induced activation of GABAA receptors by acting on distinct binding sites on the receptor complex and modulate GABA action. Due to GABA involvement in various physiological processes, many of these compounds are used for the treatment of numerous diseases (Sieghart and Sperk 2002).
By acting at GABAB receptors, GABA also achieves inhibitory effects. GABAB receptors belong to the superfamily of guanine-nucleotide-binding proteins (G proteins-coupled receptors (GPCRs)). They contain seven membrane-spanning domains with an intracellular C-terminal tail and a large extracellular N-terminal domain with ligand binding site (Bettler et al. 2004). Presynaptically located GABAB receptors are divided into auto- and heteroreceptors. The inhibition by GABAB receptors is slow and long lasting, and is mediated by indirect gating of either potassium or calcium channels via second messengers. Namely, GABAB receptors exert a presynaptic regulation of inhibitory GABA action via inhibition of presynaptically located voltage-sensitive calcium channels or at the postsynaptic site directly by alteration of synaptic vesicle priming through inhibition of adenylate cyclase and by activating inward rectifying potassium channels (Benarroch 2012). GABAB receptors are considered promising drug targets for the treatment of neurological and mental health disorders. Characteristic agonists of these receptors are baclofen (GABA analog) and 3-aminopropylphosponous acid (3-APPA; CGP27492), whereas antagonists are saclofen, phaclofen, and 2-hydroxysaclofen (Jazvinscak and Vlainic 2015).
Old nomenclature considered GABAC receptors as a distinct group, but the International Union of Basic and Clinical Pharmacology suggested these receptors to be classified as GABAA receptors class since they share high structural similarity. GABAA and GABAC receptors differ markedly in their function: GABAC receptors are more sensitive to GABA but their chloride currents are lesser and do not desensitize like those of GABAA receptors (Olsen and Sieghart 2008).
Excitatory Effect of GABA
Because of higher intracellular concentration of chloride ions inside embryonic cells, GABAergic facilitation results in chloride ions influx during development. This leads to the depolarization of the target immature neurons (Ben-Ari et al. 2007). Depolarization consequently excites the cell to fire or to activate calcium ion influx via voltage-gated channels leading to the excitatory effects of GABA. Depolarizing effects of GABA are achieved in synergy with N-methyl-d-aspartate (NMDA) receptor-mediated and voltage-gated calcium currents. GABAergic depolarizing/excitatory effect is involved in neuritic growth, cell proliferation, migration and differentiation, and synapse formation (Owens and Kriegstein 2002).
GABA in Physiology and Pathology
GABA plays a key role in the regulation of neuronal transmission throughout the brain and modulates numerous physiological and psychological processes. Thus, it affects circadian rhythms, sleep, feeding, motor control, and learning and memory. GABA is also involved in maintaining balance between excitatory and inhibitory synaptic inputs in neuronal circuits which is important not only within the neocortex but also in specific brain areas such as the hippocampus. This balance is critical for correct time and spatial processing of brain signals and is implicated in many aspects of well-being such as generation of rhythmic activity of cortical networks. Alterations in GABA levels cause misbalance between excitatory and inhibitory signals, which may trigger development and progression of various disorders. It is recognized that the excitatory-inhibitory imbalance is involved in the pathogenesis of several neurodevelopmental disorders, such as Down syndrome and autism spectrum disorders, and may also be altered in Alzheimer’s disease, anxiety disorders, etc. Enhanced activation of GABAA receptors leads to ataxia, myorelaxation, anxiolysis, sedation, hypnosis, anesthesia, and anterograde amnesia, while the decrease in receptor activity leads to increased vigilance, memory enhancement, anxiety, and seizures. The GABAergic system is therefore widely used in the treatment of anxiety disorder, insomnia, epilepsy, restlessness, and aggressive behavior (Korpi et al. 2002). Besides affecting cortical functions, GABAergic signaling plays an important role in diverse hypothalamic and brain stem circuits, and if its activity within these structures is compromised, various changes in emotional reactivity, cardiac and respiratory functions, blood pressure, food and water intake, sweating, insulin secretion, liberation of gastric acid, and motility of the colon may arise.
GABA in Non-neural Tissues
Beyond its main action within central nervous system, GABA is also present in peripheral nervous system and non-neural tissues and organs where it can be synthetized, stored, and released and able to induce different processes within the cells expressing GABA receptors. Subunits of GABAA receptors have been detected in the lung, retina, uterus, spermatozoa, adrenal medulla, pituitary lobes, kidneys, and pancreatic β-cells. Functional GABAB receptors were identified in airway epithelium. GABA may act within the cells that produce GABA (e.g., adrenal chromaffin cells) and in surrounding cells (e.g., pancreatic islet cells) (Harada et al. 2016). Released GABA may also reach adjacent organs where it can be involved in the regulation of specific functions (e.g., gastrointestinal and endocrine functions).
In recent years it has become clear that immune cells express components of the neuronal neurotransmitters systems and that there is a cross talk between nervous and immune systems. GABA function within the immune system and impact on immunomodulation have not been fully discovered yet. It has been shown that immune system may synthesize and release GABA, which can affect activation/suppression of cytokine secretion, immune cell proliferation, and migration. Thus, GABA may also have a role in autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis) (Jin et al. 2013).
Gamma-aminobutyric acid (GABA) is a small (molecular weight: 103 g/mol) zwitterionic nonprotein amino acid (Roberts and Frankel 1950) that can be found in all prokaryotic and eukaryotic organisms including plants. It is the main inhibitory neurotransmitter in the mammalian brain but has many distinct roles within different tissues in an organism. By reducing excitability of the central nervous system, GABA affects numerous physiological and psychological processes and shapes behavior. Besides acting within brain structures and peripheral neural tissue, GABA is also involved in many processes in non-neural tissues. Therefore, further investigations are necessary to reveal many roles of GABA and to address potential implications to the pathophysiology of numerous diseases. The first challenge will be to investigate GABA signaling in brain development and the mechanism by which membrane depolarization can influence developmental events, GABA effects on neuronal precursor cells and immature neurons, and later shift from excitatory to inhibitory events within GABAergic circuits. The other line of investigation is to broaden our understanding of the structure, pharmacology, and physiology of receptors that interact with GABA, as well as proteins and other molecules which regulate GABAergic neurotransmission and other functions. The detailed mechanistic understanding of GABA pathways potentially may lead to the discovery of novel therapeutic compounds that will be used to treat different diseases connected, among others, to the GABA-related disturbances.
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