Gamma-Aminobutyric Acid (GABA) (Molecular Biology)

G-Aminobutyric acid (GABA) is widely found in both vertebrates and invertebrates and in both neuronal and non-neuronal tissues. By far, its most understood and arguably most important role is as a neurotransmitter (Fig. 1). GABA is synthesised from glutamate by the enzyme glutamic acid decarboxylase (GAD). It is stored in vesicles within the synaptic terminal of neurons. When an action potential comes down the axon and reaches the synaptic terminal, calcium channels are activated, resulting in calcium influx. This leads to fusion of the neurotransmitter-containing vesicles with the cell membrane and release of the neurotransmitter, in this case GABA, into the synaptic cleft. The GABA diffuses across the synaptic gap and binds to its receptor. GABA receptors are of two types, ionotropic (GABAa) or metabotropic (GABAb), and their activation leads to hyperpolarization of the postsynaptic membrane and a decrease in the excitability of the cell. The response is terminated by desensitization of the receptors and by removal of the GABA from the synapse into the presynaptic terminal or surrounding glial cells. The latter is achieved through specific transporter proteins referred to as GABA transporters. Once taken up into a cell, two potential fates await it. The GABA can be taken up into mitochondria and metabolized to succinic semialdehyde (by GABA transaminase), and then to succinic acid, and enter the tricarboxylic acid pathway. This is referred to as the GABA shunt. Alternatively, in the presynaptic terminal, it can be recycled into synaptic vesicles and, thereby, made available for subsequent release.


Figure 1. A schematic of the vertebrate GABAergic synapse. GABA (filled circles) is generated from glutamate by glutamic acid decarboxylase (GAD). When released from presynaptic vesicles into the synaptic cleft, it diffuses across and binds to postsynaptic GABAA and GABAB receptors. It may also bind to presynaptic GABAB receptors. GABA is removed from the synaptic cleft into surrounding glial cells or the presynaptic terminal by GABA transporters. It is directly recycled into synaptic vesicles or taken up by mitochondria, converted by GABA transaminase (filled triangle) to succinic semialdehyde, and enters the tricarboxylic acid pathway.

A schematic of the vertebrate GABAergic synapse. GABA (filled circles) is generated from glutamate by glutamic acid decarboxylase (GAD). When released from presynaptic vesicles into the synaptic cleft, it diffuses across and binds to postsynaptic GABAA and GABAB receptors. It may also bind to presynaptic GABAB receptors. GABA is removed from the synaptic cleft into surrounding glial cells or the presynaptic terminal by GABA transporters. It is directly recycled into synaptic vesicles or taken up by mitochondria, converted by GABA transaminase (filled triangle) to succinic semialdehyde, and enters the tricarboxylic acid pathway.

1. Glutamic Acid Decarboxylase

GAD converts glutamic acid into GABA. In mammals there are two forms, GAD65 and GAD67 (named according to their apparent molecular weights), which are the products of two different genes (1). These two forms of GAD are expressed throughout the nervous system and also in several non-neuronal tissues including the pancreas, oviduct, and the testis. Most GABAergic neurons in the brain express both forms of GAD, but their relative abundance varies (2). GAD67 is distributed widely throughout the neuron, while GAD65 tends to be concentrated at the axon terminals (3). The role of GAD in non-neuronal tissues is, in general, poorly understood. Relatively high levels of GABA and GAD are found in the islets of Langerhans beta cells of the pancreas (4). These cells are destroyed in the autoimune disease insulin-dependent diabetes mellitus, and one of the major antigens is GAD. GAD has some amino acid sequence identity with a coxackievirus polypeptide, and it has been proposed that IDDM may result from molecular mimicry between the virus and GAD (5).

2. GABA Receptors

2.1. GABAa Receptors

GABAa receptors are ligand-gated ion channels. When GABA binds to specific recognition sites on the receptor, this triggers a conformational change leading to opening of the intrinsic anion channel allowing chloride ions to flow through into the cell. This opening of the ion channel is transient (10s of milliseconds). Two molecules of GABA must bind to open the ion channel. GABAa receptors are members of a larger ligand gated ion channel family, which contains nicotinic acetylcholine receptors, strychnine-sensitive glycine receptors, and 5-HT3 receptors. To date, the mammalian GABAa receptor gene family consists of 15 subunitstmp39-67_thumb)(6, 7). b4 and g4 have been identified in avain brain (8). Three other subunits, r1_r3, have also been identified. They are expressed primarily in the retina and have a pharmacology that differs from GABAa receptors.

As such, there are suggestions that they be classified as GABAC receptors (9). GABAa receptor subunits are 450 to 550 amino acids in length and have the structural features of the ligand-gated ion channel gene family: a putative signal peptide, a putative large extracellular domain, four putative transmembrane domains, the second of which is thought to line the pore of the channel. The subunits are classified by their relative deduced amino acid sequence homologies; within a group (eg, the a subunits), the subunits are approximately 70% homologous, whereas between groups (eg, a versus b) they are approximately 40% homologous. All the subunits have unique distributions of expression in the brain, and this expression is developmentally controlled (10). The native receptor is formed from the coassembly of the subunits in various combinations, resulting in a family of receptor subtypes. The precise subunit composition of receptor subtypes is not yet known. However, it is likely that most receptors are formed from the coassembly of a and b subunits with either a g, d, or e (or both g and q) subunit (6, 7). The receptor is a pentamer, the subunit stoichiometry of which is probably (a)2 (b)2(g) or (a)2(b)(g)2(11, 12)).

GABAA receptors are the site of action of a number of clinically important drugs including benzodiazepines (prescribed as anxiolytics, anticonvulsants, and sedatives), barbiturates, and general anaesthetics, all of which act through unique allosteric modulatory sites on the receptor, potentiating the action of GABA in opening the channel. The use of transgenic, so called "knock-in" mice has been used to delineate which GABAa receptor subtypes are responsible for the anxyiolytic properties or the sedative properties of benzodiazepines (13, 14).

GABAa receptors are also found in insects, at the neuromuscular junction and the nervous system.

The subunits that have been identified (eg, Rdl) are homologous to vertebrate GABAa receptor subunits (15). They are the sites of action of a number of insecticides, including dieldrin. Sequencing of the C. elegans genome has indicated that the family of putative GABAa receptor subunits may be quite large (16). 2.2. GABAB Receptors

These are metabotropic receptors, interacting with a guanine nucleotide binding protein (G-protein) to produce its effect, which are slower than those of the ionotropic GABAa receptor (17). Activation of the GABAb receptor can lead to inhibition (or activation) of adenyl cyclase, inhibition of voltage- gated calcium channels or activation of potassium channels (18). There is evidence that GABAb receptors can be both postsynaptic and presynaptic. The postsynaptic GABAb receptors, as compared with GABAA receptors, produce a slow and long lasting inhibition. Presynaptic receptors may act as autoreceptors, acting through a feedback mechanism to reduce the release of neurotransmitter (19). The first cDNA encoding the GABAb receptor was reported in 1997 (20) and is referred to as GABAb1. Subsequently, a homologous cDNA was identified (GABAb2), which heterodimerizes with GABAb1, and, indeed, the current consensus of opinion is that native GABAb receptors exist as such heterodimers (21). The receptor polypeptides have seven putative transmembrane domains and have significant amino acid sequence homology with metabotropic glutamate receptor gene family. There is no significant sequence homology with GABAa receptors or with other G-protein coupled receptors.

Baclofen, a GABAb receptor agonist, is used in the clinic to treat the spasticity that can result from spinal injury and also multiple sclerosis (22).

3. GABA Transporters

GABA transporters are located primarily on presynaptic terminals and surrounding glia, where they have three main functions: to regulate the concentration and duration of GABA in the synaptic cleft; to prevent the diffusion of the GABA to surrounding synapses; and to take up the GABA, thereby allowing for recycling or metabolizing. They are transmembrane proteins that use sodium and chloride ion cotransport to allow uptake of the GABA against the electrochemical gradient (23). To date, four subtypes of GABA transporter have been identified, GAT-1 (24), GAT-2 (25), GAT-3 (25), and BGT-1 (26). They are members of a larger gene family of neurotransmitter transporters including those for 5-HT, epinephrine, glycine, taurine, proline, and dopamine. The GABA transporters are approximately 600 amino acids long with 12 putative transmembrane domains and exhibit 50% to 70% amino acid sequence identity with each other. They have unique distributions in the brain, with GAT-1 being the most abundant and widespread and GAT-3 being the least abundant, with expression apparently restricted to the leptomeninges (27).

A number of compounds are known that inhibit GABA uptake by acting on GABA transporters. These include Tiagabin, which is an anti-epileptic (28).

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