Insights and progress on the biosynthesis, metabolism, and physiological functions of gamma-aminobutyric acid (GABA): a review

Introduction

Aminobutyric acid is a general term for amino acids containing amino and butyric acid groups. The main isomers are α-aminobutyric acid (AABA, also known as homoalanine), β-aminobutyric acid (BABA) and γ-aminobutyric acid (GABA, also called 4-aminobutanoic acid, piperidic acid, and piperidinic acid). Studies have shown that AABA levels are associated with depressive symptoms, tuberculosis, Reye’s syndromes, and many other diseases (Adachi et al., 2019; Oketch-Rabah et al., 2021; Wang et al., 2020b). Additionally, AABA has been studied as a general biomarker for sepsis, multi-organ failure, alcoholic liver injury and malnutrition (Wang et al., 2020b). However, BABA has not been found to be correlated with human diseases, and it is a natural product of plants controlled by their immune systems. GABA is a four-carbon non-proteinogenic water-soluble amino acid (C₄H₉NO₂) that is widely present in prokaryotes and eukaryotes in the form of free amino acid (Wang et al., 2020a; Yuan et al., 2019) (Fig. 1).

The rapid accumulation of GABA in plants is a response to abiotic stresses (such as hypoxia, heat, cold, drought, and mechanical wounding) or biotic stresses (such as wounding due to herbivory and infection) (Bown & Shelp, 2016; Wang et al., 2020b). GABA was regarded as a plant and microbial metabolite and was synthesized for the first time in 1883 (Oketch-Rabah et al., 2021). Natural GABA was first discovered in potatoes by Steward in 1949, followed by its identification in mammalian brains, and subsequently in animals as well as several other organisms, including bacteria and fungi (Steward, Thompson & Dent, 1949; Fromm, 2020; Roberts & Frankel, 1950). GABA is also a neurotransmitter found across a range of invertebrate phyla (e.g., arthropods, echinoderms, annelids, nematodes, and flatworms) (Miller, 2019). This review mainly focuses on GABA from mammals and plants. As an inhibitory neurotransmitter, GABA is most widely distributed in the central nervous system in mammals, but it is also expressed in small amounts in many peripheral tissues (e.g., liver, kidney, stomach, small intestine, and reproductive organs). GABA serves as the primary neurotransmitter in at least one-third of the neurons in the central nervous system of rats, and it accounts for nearly 30% of human brain neurons (Oketch-Rabah et al., 2021). The distribution of GABA varies across different regions of the rat brain, with the substantia nigra exhibiting the highest concentration, followed by the globus pallidus, nucleus accumbens, deep cerebellar nuclei, caudate nucleus, and cerebellar cortex (Tepper & Lee, 2007).

GABA is considered one of the inhibitory neurotransmitters involved in a variety of metabolic activities, such as depression, anxiety and stress management, hypertension, diabetes, cancer activity, oxidative, inflammatory, microbial, and allergic resistance, and protection of the liver, kidney, and intestine. As a result, it is widely used in the functional food, pharmaceutical, and agricultural products industries (Dahiya, Manuel & Nigam, 2021; Ngo & Vo, 2019; Wei et al., 2022).

In this review, we first provide a brief introduction to the three isomers of aminobutyric acid (AABA, BABA and GABA) and their functions, focusing on the discovery, development and importance of GABA. We also summarize the biosynthesis and metabolism of GABA, as well as the three GABA receptors (GABA_A, GABA_B and GABA_C) and their physiological functions in detail. Additionally, we briefly outline the role of GABA in regulating plant growth, development and stress, while placing particular emphasis on its effects on mammalian physiological functions. Subsequently, we review three commonly used methods for GABA enrichment preparation, highlighting their advantages and disadvantages. Finally, we analyze the potential of microbial fermentation for GABA enrichment and discuss the development and application of ligands for the GABA receptor binding site. New clinical trials targeting GABA for the treatment of epilepsy are briefly discussed, along with a detailed examination of potentially effective drugs or compounds targeting GABA for epilepsy treatment.

The purpose of this review is, firstly, to summarize the research progress and organize the related knowledge, allowing readers to quickly and comprehensively understand the research results concerning GABA in terms of its synthesis, metabolism, mechanism of action, receptor types, physiological functions and biosynthesis. Secondly, this review aims to attract researchers from different fields to focus on GABA research, promote communication and cooperation among neuroscience, pharmacology, botany, microbiology and other multidisciplinary disciplines, break down the boundaries between disciplines, and stimulate new research ideas and methods.

This review provides a detailed description of the three isomers of aminobutyric acid (AABA, BABA, and GABA), as well as a systematic summary of the history of GABA’s discovery and development, important milestones, and its physicochemical properties. Currently, there is a significant gap in the searchable literature concerning this section.

The main limitations of this review are as follows: First, due to the wide range of GABA-related research fields and limited space, this review only provided a brief overview of the role of GABA in regulating plant growth, development, and stress, lacking a more comprehensive and in-depth analysis. Second, some earlier references are difficult to access in their original language; however, it is worth noting that many scholars generally recognize the findings of these studies. Additionally, epilepsy, as one of the significant brain disorders, has been selected for discussion in new clinical trials targeting GABA across various conditions. On this basis, this review also discusses in depth potential drugs or compounds that target GABA, using epilepsy as an example. This will help enhance our understanding of the importance of GABA in the treatment of a wide range of diseases.

Survey/search methodology

The authors conducted a comprehensive and systematic search across PubMed, Web of Science, GenMedical, and Google Scholar. The research utilized key terms such as “Gamma-Aminobutyric Acid”, “GABA”, “neurotransmitter”, “biosynthesis”, “metabolism”, “physiological functions”, and “epilepsy”, both as subject terms and free words. The initial screening of the literature focused on titles, which was followed by a secondary screening on abstracts and keywords. In addition, ClinicalTrials.gov, Clinicaltrialsregister.eu, and the China Clinical Trials Registry were searched for ongoing studies targeting GABA for epilepsy. Ultimately, the full texts of relevant articles were obtained for further evaluation. Additionally, the authors retrieved references from pertinent studies to identify other eligible research. The target audiences for this research are diverse, comprising academics, physicians, pharmaceutical technology developers, health nutritionists, pharmaceutical producers, and innovators.

Timeline of development and key milestones to gaba

Discovery and isolation: In the 1880s, scientists succeeded in synthesizing GABA, which was thought to be a metabolite of plants and microorganisms (Oketch-Rabah et al., 2021). Natural GABA was first discovered in potatoes by Steward in 1949 (Steward, Thompson & Dent, 1949). Discoveries in the animal brain: In 1950, GABA was discovered to be present in mammalian neural tissues by Roberts & Frankel (1950), and Awapara (1950), respectively. Although the physiological activity of GABA was not yet understood, Roberts and Frankel demonstrated that GABA in mouse brains is synthesized through alpha-decarboxylation, using glutamate (Glu) as a substrate in the presence of glutamate decarboxylase (GAD) (Roberts & Frankel, 1950; Heli et al., 2022). This marked an important beginning in the study of GABA and laid the foundation for subsequent research on its role in the nervous system (Fig. 2).

Exploration as a neurotransmitter: In 1959, Takahashi et al. (1959) studied the effects of GABA on the cerebral cortex. In 1961, Curtis, Phillis & Watkins (1961) found that GABA had an inhibitory effect on spinal cord neurons, providing further evidence for GABA’s function as a neurotransmitter. In 1963, Krnjevic suggested that GABA might be a central neurotransmitter, however, this idea was not widely recognized at first (Krnjevic & Phillis, 1963). In 1967, Krnjevic and Schwartz demonstrated that GABA mimics endogenous neurotransmitters in regulating neurons in the mammalian cerebral cortex, hyperpolarizes the membrane potential, and causes a decrease in membrane resistance (Krnjevic & Schwartz, 1967).

Discovery and characterization of GABA receptors: From the 1970s to 1980s, scientists discovered and characterized GABA_A and GABA_B receptors, identifying them as ionotropic and metabotropic G protein-coupled receptors, respectively (Curtis & Johnston, 1974; Sytinsky, 1978; Bowery et al., 1980; Olsen, 1981). The molecular cloning of GABA_A receptor subunits further revealed the diversity and structure of these receptors (Schofield et al., 1987).

Follow-up researches and application expansion: After the 1980s, advances in structural biology techniques helped elucidate the detailed structure of GABA receptors and revealed the association of GABA dysfunction with various psychiatric and neurological disorders, providing important information for drug design (Mehta & Ticku, 1999; Olsen & Sieghart, 2009; Sigel & Steinmann, 2012; Miller & Aricescu, 2014; Pin & Bettler, 2016).

Since GABA was recognized as an important neurotransmitter in the 1940s, its research has progressed through multiple stages, ranging from basic biological discovery to clinical drug development. Through in-depth studies of its structural biology, scientists have identified the link between GABAergic dysfunction and numerous diseases, driving the development of GABAergic drug therapies as well as the creation and application of functional foods. In recent years, the use of new technologies has further expanded the research horizons of the GABA system, providing more possibilities for future therapeutic strategies.

Physicochemical properties of gaba

GABA has a IUPAC (International Union of Pure and Applied Chemistry) name of 4-aminobutanoic acid, a CAS (Chemical Abstracts Service) number of 56-12-2, and a UNII (Unique Ingredient Identifier) code number of 2ACZ6IPC6I (Oketch-Rabah et al., 2021). Its molecular formula is C4H9NO2, its relative molecular weight is 103.1. The amino group of GABA is located on its γ-carbon. GABA is a white to light yellow crystalline substance that can crystallize as white leaflets (in methanol-ether) or needles (in water-ethanol). It has a slightly bitter taste and exists as a zwitterion at physiological pH, possessing both positive and negative charges, with pKa values of 10.56 and 4.03, respectively (Oketch-Rabah et al., 2021; Shelp, Bown & McLean, 1999; Xu, Wei & Liu, 2017). However, GABA is not spinable. It is highly soluble in water (130 g/100 ml at 25 °C), minimally soluble in ethanol and acetone, and insoluble in cold ethanol, benzene, and ether. Its melting point is 202 °C, and it can decompose above this temperature to form pyrrolidone and water (Zhichao, 2016).

As a derivative of an amino acid, GABA not only possesses the common properties of amino acid but also exhibits unique characteristics that enable it to undergo specific chemical reactions (e.g., hydrocarbonization and acylation with chloride reactions). GABA can react with ninhydrin to form a purple compound, which has a maximum absorption at 570 nm. Additionally, GABA can react with dansyl chloride (DNS-Cl) under alkaline conditions to produce a fluorescent and stable DNS-GABA, which absorbs light in the UV region. These two reactions can be utilized for the detection of GABA (Roberts & Frankel, 1950).

Gaba biosynthesis and metabolic pathways

GABA is typically synthesized by glutamate decarboxylase (GAD; EC4.1.1.15), which utilizes pyridoxal 5′-phosphate (PLP) as a cofactor. This enzyme catalyzes the irreversible α-decarboxylation of L-glutamate to GABA, releasing CO₂ during the process (Fig. 3).

GABA shunt

The GABA shunt pathway was first described in 1970 in a study involving guinea pig cells (Balazs et al., 1970). It is named as “GABA shunt” because it bypasses two steps of the tricarboxylic acid (TCA) cycle and is associated with electron transport in the respiratory chain (Fig. 4) (Anju, Moothedath & Rema Shree, 2014). The GABA shunt is a conserved pathway in both prokaryotes and eukaryotes, and it is closely linked to the antioxidant system (Anju, Moothedath & Rema Shree, 2014). As an energy source, GABA is considered an essential defense strategy, providing NADH and/or succinate to the mitochondrial electron transport chain in the presence of impaired respiration and TCA cycle activity, as well as increased reactive oxygen species (Carillo, 2018; Carillo et al., 2019). The GABA shunt pathway is not equivalent to the conversion of a-ketoglutamate to succinate in the TCA cycle. In the TCA cycle, the metabolic conversion produces one ATP and one NADH, whereas the GABA shunt produces only one NADH. Since the GABA shunt produces less energy and only 8–10% of TCA cycle activity occurs through this shunt, its primary function is thought to be the synthesis of GABA (Patel, Balazs & Richter, 1970; Watanabe et al., 2002).

Three enzymes are involved in the GABA shunt: cytoplasmic glutamate decarboxylase (GAD), mitochondrial GABA transaminase (GABA-T), and succinate semialdehyde dehydrogenase (SSADH), respectively (Li et al., 2020; Sarasa et al., 2020). The first step in GABA anabolism involves glutamate dehydrogenase catalyzing the transamination reaction of α-ketoglutarate to produce glutamate. The second step is the irreversible decarboxylation of the alpha carbon of glutamate to GABA, catalyzed by GAD, which consumes a proton and releases CO₂. GABA catabolism begins with GABA-T catalyzing the conversion of GABA into succinic semialdehyde (SSA). SSA is then oxidized by SSADH and enters the TCA cycle as succinate (Sarasa et al., 2020; Shelp, Bown & McLean, 1999). In plants, the GABA shunt is considered necessary for maintaining the TCA cycle under both stress and non-stress conditions (Bown & Shelp, 2020). While GABA shunt enzymes are primarily located in mitochondria, their subcellular localizations vary across different organisms (GAD is located in the cytoplasmic, GABA-T and SSADH are mainly found in mitochondria, but in yeast, SSADH is located in the cytoplasm) (Cao et al., 2013; Clark et al., 2009b).

Gaba receptors

In mammals, GABA exerts its neurotransmitter effects by binding to specific receptors, which are classified as GABA_A, GABA_B, and GABA_C. These receptors differ in their sensitivities to agonists and antagonists (Bormann, 2000; Heli et al., 2022). GABA_A is an ionotropic receptor that can be activated by high concentrations of GABA, functioning as a fast-acting ligand-gated chloride channel that evokes synaptic inhibition (Adler, 2014). GABA_B receptors, on the other hand, are slow-acting metabolic G protein-coupled receptors that primarily regulate synaptic transmission and participate in various brain functions, such as recognition, learning, memory, and anxiety (Printsev, Curiel & Carraway, 2017; Wei et al., 2022). GABA_C is also an ionotropic receptor with a morphology similar to that of GABA_A receptors. In adult brains, GABA mainly acts on GABA_A and GABA_B receptors, thereby influencing the rhythmic activity generated in neural networks (Wei et al., 2022; Cuypers, Maes & Swinnen, 2018).

GABA_A receptors

The GABA_A receptor family represents a class of ligand-gated ion channels that are permeable to Cl⁻ and HCO3⁻, and have been extensively studied due to their significant physiological and therapeutic roles. GABA_A receptors consist of a heterodimeric transmembrane protein complex made up of three of the seven subunit families (α1–6, β1–3, γ1–3, δ, ε, θ, and π) (Mehta & Ticku, 1999). There is approximately 60–80% amino acid homology within each subunit class and about 30% homology between different classes (Watanabe et al., 2002). A central ion-conducting pore is formed by five adjacent subunits. Each receptor subunit has one extracellular structural domain (ECD), one transmembrane structural domain (TMD), and one intracellular structural domain (ICD) (Fig. 5A). The ECDs consist of β-sheets and contribute to agonist binding sites. The TMDs consist of pore-forming α-helices, while the structurally variable ICDs are involved in receptor assembly, transport, and aggregation.

Since the GABA_A receptor is a pentamer composed of various subunit assemblies, it should theoretically be possible to form a large number of receptor isoforms. However, the diversity of receptors is highly constrained, and mechanisms controlling the differential assembly and subcellular targeting of receptor subtypes remain unclear (Rudolph & Knoflach, 2011). Studies indicate that a functional GABA receptors contains at least one α, one β, and one γ, with the most common pentameric combinations being 2α2β1γ, 2α1β2γ, and 1α2β2γ. Among these, 2α2β1γ is the most frequently expressed in the central nervous system (Watanabe et al., 2002) (Fig. 5B).

A mature GABA_A receptor subunit consists of approximately 450 amino acid residues. Its N-terminal features a large hydrophilic extracellular structural domain (ECD), while the middle section contains four hydrophobic transmembrane structural domains (TMDs: TM1–TM4). The C-terminal contains a small extracellular domain. TM1 and TM2 are connected by a short intracellular loop, TM2 and TM3 are linked by a short extracellular loop, and TM3 and TM4 are joined by a long intracellular loop that can be phosphorylated (Ghit et al., 2021) (Fig. 5A).

The GABA binding site is located at the β+/α- junction (Watanabe et al., 2002) (Fig. 5C). In addition to the GABA binding sites, there are over 10 potential ligand binding sites that are presumably distributed at various locations on the GABA_A receptor. These sites are important drug targets and contribute to the receptor a highly complex pharmacological profile (Chua & Chebib, 2017). The GABA_A receptor family serves as the site of action for many clinically important drugs, including benzodiazepines (BZD), barbiturates, anesthetics, neurosteroids, and convulsants. These drugs can affect GABA_A receptor function by inducing or stabilizing conformational changes in the receptor, as well as binding to different metabolic or non-metabolic sites to produce physiological or clinical effects, ultimately influencing GABA_A receptor function (Mirheydari et al., 2014; Crocetti & Guerrini, 2020). The development of ligands for GABA_A receptor binding sites aims to the advancement of new therapeutic drugs.

GABA_B receptors

GABA_B is a receptor that reduces the release of norepinephrine and is insensitive to bicuculline and isoglutamate. It was first described in the 1980s by Bowery et al. (1980). Nakayasu et al. (1993) reported the purification of putative GABA_B receptor proteins for the first time. The GABA_B-Rla and GABA_B-Rlb proteins both belong to the G protein-coupled receptor family. The two are N-terminal splice variants containing 960 and 844 amino acids, respectively, and the cDNA sequences encoding both have been isolated and reported (Kaupmann et al., 1997).

The functional GABA_B receptor is known to be a specialized heterodimer formed by the co-assembly of GABA_B1 and GABA_B2 subunits, and each subunit is considered as incapable of signaling alone (Shaye et al., 2021; Xu et al., 2014). Each subunit consists of a large extracellular structural domain (Venus flytrap, VFT), a seven-transmembrane structural domain, and an intracellular C-terminus (Fritzius et al., 2022; Terunuma, 2018). Each VFT contains LB1 and LB2 structural domains or lobes, where LB1 is positioned above LB2, and the VFT can exists in either an open or closed form Frangaj & Fan (2018) (Fig. 6). The VFT is a common structural feature of all class C G protein-coupled receptors, and many selective splice variants exist for GABA_B1 (e.g., R1a, R1b, R1c, R1e, R1j, R1k, R1l, R1m, and R1n). In the human CNS, the main subtypes are R1a and R1b, which are distinguished by a pair of sushi structural domains that exist in R1a but absent in R1b. This likely relates to the presence of the sushi structural domain, which mediates multiple protein interactions of GABA_B1 (Benke et al., 1999; Terunuma, 2018). Although GABA_B2 shares 54% similarity with GABA_B1, only the VFT structural domain of GABA_B1 can bind to ligands, while the VFT of GABA_B2 cannot bind to any known ligands (Xu et al., 2014). However, the heptahelical structural domain of the GABA_B2 subunit contains binding sites for metastable regulators that can affect the affinity of ligands for binding to the GABA_B1 subunit. The interaction between the GABA_B1 and GABA_B2 subunits occurs through the coiled helical structural domain at their C-terminus (Terunuma, 2018).

GABA_B dimers, tetramers or higher order oligomers have been detected in heterologous systems. GABA_B receptors exist in an equilibrium of three aggregates (Xu et al., 2014). GABA_B receptors mediate slow and persistent inhibition by activating Galphai/o-type G proteins, leading to the liberation of Gbetagamma subunits that activate inwardly rectifying K+ channels, inactivate voltage-gated Ca²⁺ channels, or inhibit adenylate cyclase (Craig & McBain, 2014; Terunuma, 2018). The GABA analogs baclofen and 3-aminopropylphosponous acid (3APPA; CGP27492) are typical agonists of GABA_B, whereas saclofen, phaclofen and 2-hydroxysaclofen act as antagonists of GABA_B receptors (Jembrek & Vlainic, 2015).

GABA_B receptors not only mediate the actions of major inhibitory neurotransmitters but also associate with neurological and gastrointestinal disorders, as well as mental health issues. GABA_B has been identified as a multi-drug therapeutic target for conditions such as muscle spasms, pain, alcohol addiction, schizophrenia, and gastroesophageal reflux disease. As a muscle relaxant, Lioresal^® (baclofen) is the only FDA-approved drug that selectively targets GABA_B for clinical application (Shaye et al., 2020, 2021).

Physiological effects and applications of gaba

Discussions

GABA is a non-protein amino acid that occurs naturally in microorganisms, plants, and animals (Ngo & Vo, 2019). GABA is mainly synthesized through the GABA shunt and the PA pathway (Heli et al., 2022). The physiological functions of GABA differ between the animal and plant kingdoms. In plants, GABA regulates growth, development and stress responses (e.g., mechanical stress, stimulation, hypoxia, darkness, and drought). As the primary inhibitory neurotransmitter of the central nervous system, GABA is present in approximately 25–50% of mammalian neurons (Miri et al., 2023). However, the functions of GABA extend beyond the treatment of various psychiatric and neurological disorders (e.g., epilepsy treatment, anxiety suppression, depression relief, sedation and sleep assistance) to include lowering blood pressure, enhancing liver and kidney function, regulating hormone secretion, boosting immune resistance and fighting cancer, reducing asthma, controlling obesity, anti-aging and anti-skin aging, improving diabetes, and enhancing learning and memory abilities. Given GABA’s beneficial effects on human health, it has been used as a bioactive ingredient in food and clinical applications. GABA has been employed as a dietary supplement in the United States, China, Japan, and most of Europe (Tian et al., 2022). Additionally, GABA, its derivatives, and its receptors are widely utilized in clinical studies for the treatment of epilepsy, insomnia, hypertension, and stress, as well as an ergogenic substance to increase growth hormone levels (Oketch-Rabah et al., 2021; Sun & Wang, 2023; Durand et al., 2023).

GABA production methods are mainly chemical synthesis, plant enrichment, and microbial fermentation (Heli et al., 2022). GABA prepared by chemical synthesis is known to have poor safety, as it can easily be contaminated, making it unsuitable for usage in food or medicine. In contrast, GABA produced by living organisms (both plants and microorganisms) can meet the health demands of organisms with almost no side effects. Consequently, research on GABA-rich plants and microorganisms has become a hot topic, with many researchers focusing on the development of GABA-rich functional foods. Among these methods, microbial fermentation is preferred for commercial use because it is more effective, less costly, and more environmentally friendly. Currently, although both bacteria and fungi serve as good sources for GABA production, the genus Lactobacillus has been the most widely used for this purpose (Oketch-Rabah et al., 2021).

GABA interacts with three types of receptors: GABA_A, GABA_B and GABA_C (Bormann, 2000). GABA_A and GABA_C are fast-acting ligand-gated chloride channel receptors, while GABA_B is a slow-acting metabolic G protein-coupled receptor (Kim et al., 2018). Each receptor possesses different binding sites, thus leading to distinct pharmacological and physiological characteristics. The development of ligands for the GABA receptor binding site has contributed to a better understanding of various physiological functions and pathological processes, as well as to the development of new therapeutic drugs.

Epilepsy is a prevalent chronic brain disease characterized by recurrent seizures (Chen et al., 2023). These seizures are caused by abnormal discharges of neurons in the brain, which are transient and sudden, that can occur at any age and hence affect individuals throughout their lifespan (Chen et al., 2024). GABA plays a crucial role in the treatment of epilepsy and can effectively control seizures by regulating neuronal activity. A clinical trial assessing the effects of GABA on epilepsy and ADHD in children is currently in Phase IV (NCT04144439). Gabapentin and pregabalin, which are structurally similar to GABA, do not act directly on GABA receptors. Instead, these two drugs are used primarily for the treatment of partial-onset epilepsy by binding to the α2δ subunit of voltage-gated calcium channels, which reduces the release of excitatory neurotransmitters and indirectly enhances GABAergic inhibition. Both drugs have completed phase III clinical trials (NCT00603473; NCT00448916). Pregabalin has been approved by the U.S. Food and Drug Administration (FDA) as an adjunctive treatment for partial-onset seizures in adults (Cross, Viswanath & Sherman, 2024). Sodium valproate has a molecular structure similar to that of the neurotransmitter GABA, and the addition of lamotrigine to valproate is currently being investigated to evaluate its effects on epilepsy in children, with clinical trials in phase IV (NCT05881928). ETX101 consists of a non-replicating recombinant adeno-associated virus serotype 9 vector designed for the delivery of a GABAergic regulatory element and an engineered transcription factor capable of enhancing the transcription of the SCN1A gene. ETX101 has shown promising results in clinical epilepsy studies and is currently undergoing clinical trials (Lersch et al., 2023; NCT06112275). Muscimol, which resembles a natural brain chemical called GABA, has been shown as capable of reducing seizures in rats. A clinical trial is currently in phase I to assess the safety and efficacy of injecting muscimol into the brain to control refractory epilepsy (i.e., seizures that are frequent and persist despite treatment) (NCT00005925).

There are many potential possible drugs or compounds that could target GABA for the treatment of epilepsy. Recent research has focused on epilepsy associated with disorders such as Tuberous Sclerosis Complex (TSC), Lennox-Gastaut syndrome, focal refractory epilepsy, and Dravet syndrome. GABA_A receptors play a key role in inhibitory neurotransmission; therefore, drugs targeting GABA_A receptors have received extensive research attentions in the treatment of epilepsy. GABA_A receptor modulators can be categorized into benzodiazepines and non-benzodiazepines.

Representative drugs of the benzodiazepine class include clobazam, clonazepam, and diazepam. These are three drugs share similarities in the mechanisms for treating epilepsy: they can bind to benzodiazepine receptors, promoting the binding of GABA to GABA receptors. This process increases the frequency of chloride channel openings, leading to hyperpolarization of the neuronal membrane and inhibition of neuronal firing. By enhancing the inhibitory effects of GABA, these drugs can reduce neuronal excitability and exert an antiepileptic effect. Clobazam is an oral 1,5-benzodiazepine that enhances GABA_A receptor function and reduces the frequency and severity of seizure, completed a Phase IV clinical trial in 2018 (NCT02726919). It is used globally for the treatment of many types of epilepsy and is approved in the United States for the treatment of Lennox-Gastaut syndrome (Gauthier & Mattson, 2015). Clonazepam orally disintegrating tablets (Klonopin^®) have been used in the acute treatment of epileptic seizures and are indicated for both seizures and panic attacks. More than 50% of seizures stop within 10 min, and it is as effective as diazepam rectal gel (Penovich et al., 2024; Troester, Hastriter & Ng, 2010; Cloyd et al., 2021). Diazepam rectal gel was approved by the U.S. Food and Drug Administration in 1997 for patients with acute epilepsy aged 2 years and older. However, rectal administration has limitations. Diazepam nasal spray was also approved in 2020 for the treatment of patients with acute epilepsy aged 6 years and older (Penovich et al., 2024).

Non-benzodiazepines, such as phenobarbital, ganaxolone, and darigabat, also target GABA_A receptors. Phenobarbital effectively prolongs the opening time of the chloride channel by acting on the GABA_A receptor, significantly enhancing the inhibitory effect of GABA. It can effectively prevent subsequent seizures through intravenous administration and has been used to treat neonatal epilepsy, having already completed phase III clinical trials (NCT04320940). Ganaxolone is a neuroactive steroid that positively allosterically modulates GABA_A receptors, effectively reducing seizures, with a mechanism of action similar to that of phenobarbital. Currently, ganaxolone is in phase II clinical trials, showing potential therapeutic value for TSC (NCT04285346). Darigabat, a positive allosteric modulator of GABA_A receptors, has demonstrated significant efficacy in a mesial temporal lobe epilepsy (a preclinical model of medically refractory focal epilepsy). A Phase II clinical trial (NCT04244175) is currently underway to evaluate the efficacy and safety of daligabat in patients with medically refractory focal seizures (Gurrell et al., 2022).

In addition to research on drugs targeting the GABA_A receptor for the prevention and treatment of epilepsy, research is actively underway on a wide range of drugs or compounds targeting the GABA_A, GABA_B and GABA_C receptors for the prevention and treatment of a variety of disorders, such as Parkinson’s disease, muscle spasms, depression and others. These studies are aimed at developing more effective drugs with fewer side effects, with a view to providing a wider range of therapeutic options in clinical applications.

Since the interaction between GABA receptors and ligands can effectively combat a variety of diseases and facilitate pharmacological research and drug development. It has become a major focus of research. However, the mechanism by which GABA controls the differential assembly of receptor subtypes and subcellular targeting remains unclear. The number, location, and roles of GABA binding sites have not yet been systematically and thoroughly investigated. The study of the multiple physiological functions of GABA continue to be the primary focus of current research. Additionally, the development of GABA-enriched functional foods has emerged as a hot research topic. This research primarily involves the development of high GABA-producing strains, improving the stability of GABA during storage, and developing new enrichment techniques without compromising the quality of food or other active ingredients. Currently, the microbial fermentation method for GABA production holds broad prospects, with bacteria, fungi and engineered bacteria being widely studied. An increasing number of studies on GABA will provide more solutions for drug development and disease treatment.

Conclusions

GABA is a naturally occurring non-protein amino acid found in microorganisms, plants and animals, synthesized through various pathways. It exhibits different physiological functions in both the animal and plant kingdoms and provides numerous benefits for humans. The primary methods for GABA production include chemical synthesis, plant enrichment, and microbial fermentation. Among these, microbial fermentation has become the preferred choice for commercial production due to its high efficiency, low cost, and reduced environmental impact. In terms of physiological mechanisms, GABA interacts with three types of receptors: GABA_A, GABA_B and GABA_C. The development of ligands targeting the binding sites of these receptors not only contributes to in-depth studies of related diseases but also facilitates the research and development of new drugs. In the field of epilepsy treatment, GABA plays a critical role, and various drugs are available that control seizures by targeting the GABA system through different mechanisms. Clinical trials related to these drugs are at various stages, indicating promising prospects for development. While the interaction between GABA receptors and ligands has become a hot research topic, there is still a need for in-depth exploration of how GABA controls subtype assembly, subcellular targeting mechanisms, and the specific functions of binding sites. Additionally, the development of GABA-rich functional foods is emerging as a key area of research, with the microbial fermentation method showing great promise in this field. In conclusion, GABA is expected to have many positive impacts on health, daily life and industrial development across various fields, including medicine, food, agriculture, and cosmetics.