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Introduction to SLC6 transporters The solute carrier (SLC) 6 family of membrane proteins perform the Na+-coupled symport of amino acids and amino torin mg derivatives into cells throughout the body. Disrupting neurotransmitter SLC6-mediated transport leads to a plethora of neurological disorders including autism, anxiety, depression, epilepsy, Parkinson’s disease, schizophrenia and pain . SLC6 neurotransmitter transporters are located at the synaptic cleft, with transporter-specific distributions in either the pre-synaptic neuronal membrane, or the glial cell membranes. Transporters in the presynaptic membrane remove substrate from the synaptic cleft and recycle it back into the presynaptic neuron. These are the dopamine (DAT), noradrenaline (NET), serotonin (SERT), type 2 glycine (GlyT2) and type 1 GABA transporters (GAT1). In contrast, the type 1 glycine (GlyT1) and type 3 GABA transporters (GAT3) are located in glial cell membranes, where they remove substrate from the cleft. The presynaptic and glial membranes contain distinct lipid assemblies whose role in SLC6-mediated transport is poorly understood. While universally treating SLC6-linked neurological disorders represents a significant medical challenge, these transporters share many salient features. All SLC6 transporters characterised to date have a conserved structural architecture and substrate transport mechanism, common to other excitatory amino acid transporters in the SLC superfamily. SLC6 transporters are comprised of 600–700 amino acids, arranged into 12 transmembrane helices, with both the N- and C-termini located intracellularly (Fig. 1A). Substrates are transported via a secondary active transport mechanism, where a molecule of substrate is co-transported with Na+ and Cl− down the Na+ electrochemical gradient. The precise stoichiometry of substrate:Na+:Cl− transport is specific for each type of SLC6 transporter. Crystal structures of SLC6 transporters and their homologues indicate that substrate transport occurs via an alternating access mechanism, wherein the protein cycles through a series of conformational states while being accessible to substrates from one side of the membrane at any point in time [2,3]. In this mechanism, substrates in the synaptic cleft bind when the transporter adopts a conformation that exposes the binding site to the extracellular solution. This induces a series of conformational changes that occlude the extracellular access pathway to the substrate binding site, while opening the cytoplasmic access pathway, and allows substrate to be released intracellularly [4,5].
SLC6 binding and transport mechanisms Crystal structures of sodium-coupled amino acid transporters have provided clues to the binding and transport mechanism that these proteins use. Direct structural insights have only been available since 2005, when the first crystal structure of a sodium-coupled small amino acid transporter, LeuT (PDB ID 2A65) , was elucidated. Although LeuT is a prokaryotic transporter, its structure, combined with biochemical, mutagenesis, and homology modelling data, directed our understanding of eukaryotic SLC6 transporters. This understanding has been supplemented by the recently-solved crystal structures of eukaryotic transporters for dopamine (DAT) (PDB ID 4M48) in 2013 , and serotonin (SERT) (PDB ID 5I6X) in 2016 . Collectively, they have provided the structural details of the substrate binding site, S1, and the location of two conserved Na+ binding sites, the Na1 and Na2 site. Figs. Figure 1B and Figure 2 show the S1 site is located near the centre of the protein. It contains a polar region (formed by side chains from TM 1 and 6) that bind substrate α-amino and α-carboxylate groups, and a hydrophobic pocket (formed by side chains from TM1, TM3 and TM6) that binds hydrophobic substrate side chains. The particular amino acid composition of the S1 site, and the resultant volume and shape of the S1 cavity are believed to dictate its substrate specificity. A second putative substrate binding site, S2, has been identified in LeuT, DAT and NET [, , , ]. The S2 site is located along the extracellular pathway, between the S1 site and the extracellular space .