2.A.23 The Dicarboxylate/Amino Acid:Cation (Na+ or H+) Symporter (DAACS) Family
The members of the DAACS family catalyze Na+ and/or H+ symport together with (a) a Krebs cycle dicarboxylate (malate, succinate, or fumarate), (b) a dicarboxylic amino acid (glutamate or aspartate), (c) a small, semipolar, neutral amino acid (Ala, Ser, Cys, Thr), (d) both neutral and acidic amino acids or (e) most zwitterionic and dibasic amino acids. The bacterial members are of about 450 (420-491) amino acyl residues while the mammalian proteins are of about 550 (503-574) residues in length. These proteins possess between ten and twelve hydrophobic segments per polypeptide chain. Two of them, human EAAT2 (TC #2.A.23.2.2) and E. coli GltP (TC #2.A.23.1.1) have been shown to be homotrimers (Gendreau et al., 2004). A specific topological model in which 7 α-helical TMSs are followed by a reentrant loop-pore structure followed by one final TMS is presented in Slotboom et al. (1999) and Leighton et al. (2002). Possibly, the transporter consists of eight TMSs, and one or two pore-loop structures that dip into the membrane (one between TMSs 6 and 7, the other between TMSs 7 and 8) in a fashion reminiscent of pore-loop structures found in VIC family ion channels (TC#1.A.1) (Grunewald et al., 2002). This family of transporters has been reviewed (Grewer et al. 2013). Functional up-regulation of GluTs may provide a pharmacotherapeutic approach for the management of chronic pain using pyridazine derivatives and beta-lactams (Gegelashvili and Bjerrum 2019).
All of the bacterial proteins cluster together on the phylogenetic tree as do the mammalian proteins. The mammalian permeases that transport neutral amino acids cluster separately from those that are specific for the acidic amino acids. Among the mammalian proteins are neuronal excitatory amino acid neurotransmitter permeases. One of these (the GLT-1 L-glutamate/L-aspartate/D-aspartate transporter) has been shown to cotransport the neurotransmitter with 3 Na+ and 1 H+ and to countertransport 1 K+. The EAAT3 carrier (also called the EAAC1 carrier) uses Arg-447 to bind dicarboxylic amino acids in the presence of K+ but not monocarboxylic amino acids (Bendahan et al., 2001). Larsson et al. (2010) have identified the 3rd Na+ binding site and provided evidence for the mechanism of transport. Glutamate and Na+ binding activates an uncoupled chloride conductance in EAAT proteins, showing that they can function both as carriers and channels, and the two functions may arise from separate transmembrane domains (Ryan and Vandenberg 2006).
Some members of the DAACS family from animals, such as EAAT1, EAAT2, EAAT3 and EAAT4, can apparently be induced to function in a 'channel mode' wherein the transporter allows ion passage without being coupled to substrate translocation. This effect may involve a chloride-permeable, anion-selective channel. Some evidence suggests that the N- and C-termini of EAAT3 as well as two histidyl residues (in EAAT4) in the extracellular loop between TMSs 3 and 4 play a role in conversion to the channel mode (Li et al., 2000). The loop between TMSs 3 and 4 functions to allow regulation of this current by Zn2+ (Mitrovic et al., 2001). Distinct conformational states mediate carrier versus channel function, and a dynamic equilibrium exists between the two forms (Borre et al., 2002; Ryan et al., 2002). It is possible to isolate anion permeability mutants in TMS2 that show no change in glutamate transport (Ryan et al., 2004). EAAT4 but not EAAT2 anion channels display voltage-dependent gating that is modified by glutamate (Melzer et al., 2003). Possibly the channel activity is related to their trimeric structures (Gendreau et al., 2004). Torres-Salazar and Fahlke (2007) have reported that neuronal glutamate transporters (EAATs) vary in substrate transport rate but not in unitary anion channel conductance.
The 3-D structure of a member of the DAACS family has been determined (Boudker et al., 2007; Yernool et al., 2004) (see 2.A.23.1.5). The putative transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. Each protomer harbors 8 TMSs and two reentrant helical hairpins. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. There are 3 independent translocation pathways. The first six transmembrane segments form a distorted 'amino-terminal cylinder' and provide all interprotomer contacts, whereas transmembrane segments TM7 and TM8, together with hairpins HP1 and HP2, coalesce to form a highly conserved core within the amino-terminal cylinder. It is proposed that transport of aspartate or glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane. Helical hairpin 2 is the extracellular gate that controls access of aspartate and the ions to the internal binding site (Boudker et al., 2007). Molecular simulations have provided evidence for the substrate translocation pathway (Gu et al., 2009). The central cavity in trimeric glutamate transporters restricts ligand diffusion (Leary et al., 2011).
Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na+/H+/K+ transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. Machtens et al. 2015 examined the prokaryotic homolog GltPh (TC# 2.A.23.1.5) and mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels (Machtens et al. 2015).
As noted above, EAATs couple the transport of glutamate to the co-transport of three Na+ ions and one H+ ion into the cell, and the counter-transport of one K+ ion out of the cell. The EAAT Cl- channel is activated by the binding of glutamate and Na+, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport (Qu et al. 2019).Several O-Benzylated l-threo-beta-hydroxyaspartate derivatives have been developed as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino 3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as a low-nanomolar inhibitor (Leuenberger et al. 2016).
The generalized transport reaction catalyzed by members of the DAACS family is:
substrate (dicarboxylate or amino acid) (out) + 4 M+ [M+ =1 H+ and 3 Na+] (out) + K+ (in) →
substrate (in) + 4M+ (in) +K+ (out)