International Journal of Biological Macromolecules 158 (2020) 750–772 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac γ-Aminobutyric acid transporters as relevant biological target: Their function, structure, inhibitors and role in the therapy of different diseases Kamil Łątka, Jakub Jończyk, Marek Bajda ⁎ Jagiellonian University Medical College, Faculty of Pharmacy, Department of Physicochemical Drug Analysis, 30-688 Cracow, Medyczna 9, Poland article info abstract Article history: γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the nervous system. It plays a crucial role Received 3 January 2020 in many physiological processes. Upon release from the presynaptic element, it is removed from the synaptic cleft Received in revised form 17 April 2020 by reuptake due to the action of GABA transporters (GATs). GATs belong to a large SLC6 protein family whose Accepted 18 April 2020 characteristic feature is sodium-dependent relocation of neurotransmitters through the cell membrane. GABA Available online 1 May 2020 transporters are characterized in many contexts, but their spatial structure is not fully known. They are divided Keywords: into four types, which differ in occurrence and role. Herein, the special attention was paid to these transporting GABA transporters proteins. This comprehensive review presents the current knowledge about GABA transporters. Their distribu- Function tion in the body, physiological functions and possible utilization in the therapy of different diseases were fully Structure discussed. The important structural features were described based on published data, including sequence analy- Inhibitors sis, mutagenesis studies, and comparison with known SLC6 transporters for leucine (LeuT), dopamine (DAT) and Therapy serotonin (SERT). Moreover, the most important inhibitors of GABA transporters of various basic scaffolds, di- verse selectivity and potency were presented. © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). 1. Introduction sequentially: GAT-1, GAT-2, GAT-3, and GAT-4, while rat and human transporters are designated as follows: GAT-1, BGT-1, GAT-2, and γ-Aminobutyric acid (GABA) is the primary inhibitory neurotrans- GAT- 3. All four proteins belong to one family of sodium-dependent mitter in mammalians [1]. It is thought that GABA is prevalent in membrane transporters (SLC6) [6,8]. The SLC6 family is one of the larg- about 30% of all synapses in the brain [2]. It plays many functions, pro- est solute carrier (SLC) families, containing highly similar 20 transporter viding anticonvulsant, anxiolytic, muscle relaxing, and sedative effects. proteins. It is divided into four subclasses: monoamine, GABA, amino GABA is produced in GABAergic system neurons from glutamic acid by acid and orphan transporters, based on sequence similarity and sub- the action of glutamic acid decarboxylase (GAD). Further, GABA is col- strate specificity (Fig. 1)[9–12]. The whole family includes transporters lected in synaptic vesicles by the vesicular GABA transporter (VGAT) for norepinephrine, dopamine, serotonin, GABA, and glycine. The ma- [3]. The depolarization of presynaptic neurons stimulates the release jority of transporters for neurotransmitters contain approximately 600 of γ-aminobutyric acid from axon terminals. GABA acts as an agonist amino acid residues, ranging from 599 for GAT-1 to 632 for SERT [13]. on three types of membrane receptors: GABA-A, GABA-B and GABA-C Only two glycine transporters are exceptions and contain approxi- [4]. GABA-A and GABA-C receptors belong to the ionotropic class of re- mately 700 residues. The various types of transporters occur in the dif- ceptors and are ligand-gated chloride channels. GABA-B is a metabotro- ferent structures of the nervous system [8,12]. They are widely pic receptor, i.e. it is coupled with G-protein. Finally, GABA is disposed of distributed on neurons and less frequently on glial cells. Dysfunction in two ways: reuptake or metabolism [5]. Reuptake is dominant and oc- of GABAergic system and consequently reduction of the inhibitory effect curs through the operation of GABA transporters (GATs) [6]. On the of gamma-aminobutyric acid can lead to a variety of pathological pro- other hand, the metabolism of GABA by transaminase leads to the inac- cesses and the occurrence of diseases, such as anxiety disorders, epi- tivation of the neurotransmitter. GABA transporters are divided into lepsy, neurodegenerative disorders, schizophrenia, insomnia, motion four types, and their nomenclature is different depending on the organ- impairment or pain states [14,15]. Therefore, the ability to potentiate ism in which they occur [7]. In mice, GABA transporters are numbered the GABA effect by inhibiting the reuptake is an attractive therapeutic target. Our comprehensive review aims to gather all current knowledge about GABA transporters. The presented data contain both basic infor- ⁎ Corresponding author. mation as well as recent achievements in the GAT field. Their distribu- E-mail address: [email protected] (M. Bajda). tion in the body, functions, structure, therapeutic potential and https://doi.org/10.1016/j.ijbiomac.2020.04.126 0141-8130/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 751 Fig. 1. Phylogenetic tree for SLC6 family of sodium-neurotransmitter symporters [8]. inhibitors are described and discussed. Special attention is paid to struc- neurotransmission, the primary function of all GABA transporters is to tural aspects as they are essential for the ligand binding process. Such a bind the extracellular GABA and transport it into the cytoplasm. In this wide range of topics makes review useful for researchers interested in way, GABA transporters regulate the level of GABA at the synaptic GABA transporters: both pharmacologists and medicinal chemists cleft [16–19]. The GABA molecules that reach the GABA receptors from looking for novel active compounds. the synaptic space induce postsynaptic inhibitory potential (a phase known as the inhibition of postsynaptic current (IPSC)). The GABA con- 2. The distribution and physiological functions of GABA transporters centration continues to increase until the late IPSC decay phase. Another activity of GABAergic conduction that is influenced by GABA uptake is Gamma-aminobutyric acid (GABA) transporters are heteroge- the tonic (permanent) stimulation of GABA receptors [20]. This stimula- neously distributed in various areas of the central nervous system tion is a result of the constant maintenance of the low concentration of (CNS) and many vital organs (Fig. 2). In the context of GABAergic GABA in the extracellular space. GABA transporters capture the released Fig. 2. Anatomical and cellular localization of GABA transporters [12,21–25]. 752 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 neurotransmitter until its concentration reaches the level that is neces- for the neuronal release [18]. In this way, GAT-3 limits the prolonged re- sary to stimulate the primary pool of receptors. Maintaining a constant lease of GABA by neurons. GAT-3 was also found expressed on astro- tonic inhibitory potential facilitates reducing the ascending excitatory cytes distant from GABAergic terminals and occasionally on stimuli and lowering the pulsation resulting from continuous depolari- neighboring axon terminals near the excitatory synapses, where GABA zation. The diverse distribution of different GABA transporters in the uptake by GAT-3 could limit the action of GABA on distant GABA recep- brain, in combination with their different affinities to GABA, results in tors [41,56]. an individualized response of neurons to the release of the Differences in the cellular distribution of GAT-1 and GAT-3 allow neurotransmitter. both these transporters to perform different roles in synaptic function. GAT-1 limits the escape of GABA from the synaptic cleft, while GAT-3 2.1. GAT-1 and GAT-3 controls the tonic inhibition by mediating the basic concentration of GABA [6,57]. Both transporters complement each other's functions by The distribution and function of the GAT-1 and GAT-3 transporters regulating different signaling pathways mediated by GABA released are related to each other. Both GAT-1 and GAT-3 are expressed mostly via vesicular and nonvesicular mechanisms or during low-frequency in the CNS [25–32]. Distribution of GAT-1 is strongly related to the local- and sustained neuronal activity [58,59]. The amplitude and duration of ization of GABAergic neurons [17,18]. Presynaptic localization of GAT-1 the release of GABA into the synaptic cleft affect the ability of GABA was proved using in situ hybridization and immunocytochemistry in transporters to alter the receptor activation. GABA uptake exerts only GABAergic neurons [27,33–35]. The highest level of GAT-1 expression a small effect on the IPSC profile after a brief increase of transmitter con- was detected in the cerebral cortex in symmetrical synaptic axon termi- centration, but it significantly regulates the recruitment of neighboring nals in the rat brain [26,36]. Experiments on the cerebral cortex of synapses [60–63]. GABA transporters limit the escape