Glutamate (excitatory amino acid) transporters

Overview: Plasma membrane located glutamate transporters (nomenclature proposed by Amara and Arriza, 1993) are members of the solute carrier family 1 (SLC1) of sodium-dependent transporters that also includes the neutral amino acid transporters ASCT1 and ASCT2 (Palacín et al., 1998; Kanai and Hediger, 2003; 2004; Beart and O'Shea, 2007). Glutamate transporters present the unusual structural motif of 8TM segments and 2 re-entrant loops (Grunwald and Kanner, 2000). The crystal structure of a glutamate transporter homologue (Glt_Ph) from Pyrococcus horikoshi i supports this topology and indicates that the transporter assembles as a trimer, where each monomer is a functional unit capable of substrate permeation (Yernool et al., 2004; Boudker et al., 2007). These structural data are in agreement with the proposed quaternary structure for EAAT2 (Gendreau et al., 2004) and several functional studies that propose the monomer is the functional unit (Ryan et al., 2004; Grewer et al., 2005; Koch et al., 2007; Leary et al., 2007). Splice variants of EAAT2 have recently been shown to form homomeric and heteromeric assemblies at the cell surface (Peacey et al., 2009). The activity of glutamate transporters located upon both neurones (predominantly EAAT3, 4 and 5) and glia (predominantly EAAT 1 and 2) serves, dependent upon their location, to regulate excitatory neurotransmission, maintain low ambient extracellular concentrations of glutamate (protecting against excitotoxicity) and provide glutamate for metabolism including the glutamate-glutamine cycle. The Na⁺/K⁺/ATPase that drives transport has been demonstrated to co-assemble with EAAT1 and EAAT2 (Rose et al., 2009). Recent evidence supports novel roles in brain for splice variants of EAAT1 and EAAT2 (Macnab and Pow, 2007; Sullivan et al., 2007). Enhanced expression of EAAT2 resulting from administration of ß-lactam antibiotics (e.g. ceftriaxone), or the neuroimmunophilin GPI-1046, is neuroprotective (Rothstein et al., 2005; Ganel et al., 2006). Enhanced expression by ceftriaxone has been proposed to occur through NF-kappaB-mediated EAAT2 promoter activation (Lee et al., 2008), although the protective affects of ceftriaxone in a mouse model of multiple sclerosis are thought to mediated by a reduction in T cell activation with no effect on EAAT2 expression, or function (Melzer et al., 2008). A thermodynamically uncoupled Cl^- flux, activated by Na⁺ and glutamate (Kanner and Borre, 2002; Kanai and Hediger, 2003; Grewer and Rauen, 2005) (or aspartate in the case of Glt_Ph, Ryan and Mindell, 2007), is sufficiently large, in the instances of EAAT4 and EAAT5, to influence neuronal excitability (Veruki et al., 2006). In the kidney, EAAT3 located in the apical membrane of proximal tubular cells is responsible for the reabsorption of glutamate (Hediger, 1999). Three structurally and functionally distinct vesicular glutamate transporters (VGLUT1, 2 and 3) of the SLC17 family are responsible for concentrating glutamate within synaptic vesicles (Reimer and Edwards, 2004).

Nomenclature	EAAT1	EAAT2	EAAT3
Other names	GLAST, SLC1A3	GLT1, SLC1A2	EAAC1, SLC1A1
Ensembl ID	ENSG000000079215	ENSG00000110436	ENSG00000106688
Endogenous substrates	L-glutamate, L-aspartate	L-glutamate, L-aspartate	L-glutamate, L-aspartate
Inhibitors (KB or Ki)	UCPH-101 (IC50= 120 nM – membrane potential assay, Jensen et al. (2009), DL-TBOA (9 µM)	WAY-213613 (IC50= 130 nM), DL-TBOA (0.12 µM), (2S,4R)-4-methylglutamate (3.4 µM), dihydrokainate (9 µM), Threo-3-methylglutamate (18 µM)	NBI-59159 (IC50= 25 nM), DL-TBOA (IC50= 8 µM), L-β-BA (IC50= 0.8 µM –[3H]-D-aspartate uptake assay)
Probes	[3H]-ETB-TBOA (KD= 15.5 nM), [3H]-[(2S,4R)-4-methylglutamate, [3H]-D-aspartate, [3H]-L-aspartate	[3H]-ETB-TBOA (KD= 16.2 nM), [3H]-[(2S,4R)-4-methylglutamate, [3H]-D-aspartate, [3H]-L-aspartate	[3H]-ETB-TBOA (KD= 320 nM), [3H]-D-aspartate, [3H]-L-aspartate
Stoichiometry	–	3Na+: 1H+: 1glutamate (in): 1K+ (out)	3Na+: 1H+: 1glutamate (in): 1K+ (out)

Nomenclature	EAAT4	EAAT5
Other names	SLC1A6	SLC1A7
Ensembl ID	ENSG00000105143	ENSG00000162383
Endogenous substrates	L-glutamate, L-aspartate	L-glutamate, L-aspartate
Inhibitors (KB or Ki)	DL-TBOA (4.4 µM), Threo-3-methylglutamate (50 µM)	DL-TBOA (3.2 µM)
Probes	[3H]-ETB-TBOA (KD= 24.8 nM), [3H]-D-aspartate, [3H]-L-aspartate	[3H]-ETB-TBOA (KD= 29.5 nM), [3H]-D-aspartate, [3H]-L-aspartate

The K_B (or K_i) values reported, unless indicated otherwise, are derived from transporter currents mediated by EAATs expressed in voltage-clamped Xenopus laevis oocytes (Vandenberg et al., 1997; Shimamoto et al., 1998; Eliasof et al., 2001; Shigeri et al., 2001). K_B (or K_i) values derived in uptake assays are generally higher (e.g. Shimamoto et al., 1998). In addition to acting as a poorly transportable inhibitor of EAAT2, (2S,4R)-4-methylglutamate, also known as SYM2081, is a competitive substrate for EAAT1 (K_M= 54µM; Vandenberg et al., 1997; Huang et al., 2009) and additionally is a potent kainate receptor agonist (Zhou et al., 1997), which renders the compound unsuitable for autoradiographic localisation of EAATs (Apricòet al., 2007). Similarly, at concentrations that inhibit EAAT2, dihydrokainate binds to kainate receptors (Shimamoto et al., 1998). WAY-855 and WAY-213613 are both non-substrate inhibitors with a preference for EAAT2 over EAAT3 and EAAT1 (Dunlop et al., 2003; Dunlop et al., 2005). NBI-59159 is a non-substrate inhibitor with modest selectivity for EAAT3 over EAAT1 (>10-fold) and EAAT2 (fivefold) (Coon et al., 2004; Dunlop, 2006). Analogously, L-β-threo-benzyl-aspartate (L-β-BA) is a competitive non-substrate inhibitor that preferentially blocks EAAT3 versus EAAT1, or EAAT2 (Esslinger et al., 2005). [³H]-[(2S,4R)-4-methylglutamate demonstrates low affinity binding (K_D≅ 6.0 µM) to EAAT1 and EAAT2 in rat brain homogenates (Apricòet al., 2001) and EAAT1 in murine astrocyte membranes (Apricòet al., 2004), whereas [³H]-ETB-TBOA binds with high affinity to all EAATs other than EAAT3 (Shimamoto et al., 2007). The novel isoxazole derivative (-)-HIP-A may interact at the same site as TBOA and preferentially inhibit reverse transport of glutamate (Colleoni et al., 2008). Threo-3-methylglutamate induces substrate-like currents at EAAT4, but does not elicit heteroexchange of [³H]-aspartate in synaptosome preparations, inconsistent with the behaviour of a substrate inhibitor (Eliasof et al., 2001). Parawixin1, a compound isolated from the venom from the spider Parawixia bistriata is a selective enhancer of the glutamate uptake through EAAT2 but not through EAAT1 or EAAT3 (Fontana et al., 2003; 2007;). In addition to the agents listed in the table, DL-threo-β-hydroxyaspartate and L-trans-2,4-pyrolidine dicarboxylate act as non-selective competitive substrate inhibitors of all EAATs. Zn²⁺ and arachidonic acid are putative endogenous modulators of EAATs with actions that differ across transporter subtypes (reviewed by Vandenberg et al., 2004).

Glossary

Abbreviations:

DL-TBOA

DL-threo-β-benzyloxyaspartate

GPI-1046

(3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate

ETB-TBOA

(2S, 3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate

(-)-HIP-A

(-)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic acid

NBI-59159 (also known as WAY-209429)

(N-4-(9H-fluoren-2-yl)-L-asparagine

WAY-855

3-amino-tricyclo[2.2.1.0^2.6]heptane-1,3-dicarboxylic acid

WAY-213613

N(4)-[4-(2-bromo-4,5-difluorophenoxy)phenyl]-L-asparagine

UCPH-101

2-amino-4-(4-methoxyphenyl)-7-(naphthalene-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile

Further Reading

Beart PM, O'Shea RD (2007). Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement. Br J Pharmacol150: 5–17

Bridges RJ, Esslinger CS (2005). The excitatory amino acid transporters: pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes. Pharmacol Ther107: 271–285.

Bunch L, Erichsen MN, Jensen AA (2009). Excitatory amino acid transporters as potential drug targets. Expert Opin Ther Targets13: 719–731.

Danbolt NC (2001). Glutamate uptake. Prog Neurobiol65: 1–105.

Dunlop J (2006). Glutamate based therapeutic approaches: targeting the glutamate transport system. Curr Opin Pharmacol6: 103–107.

Dunlop J, Butera JA (2006). Ligands targeting the excitatory amino acid transporters (EAATs). Curr Top Med Chem6: 1897–1906.

Grewer C, Gameiro A, Zhang Z, Tao Z, Braams S, Rauen T (2008). Glutamate forward and reverse transport: from molecular mechanism to transporter-mediated release after ischemia. IUBMB Life60: 609–619.

Grewer C, Rauen T (2005). Electrogenic glutamate transporters in the CNS: molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling. J Membr Biol203: 1–20.

Hediger MA (1999). Glutamate transporters in kidney and brain. Am J Physiol277: F487–F492.

Hinoi E, Takarada T, Tsuchihashi Y, Yoneda Y (2005). Glutamate transporters as drug targets. Curr Drug Targets CNS Neurol Disord4: 211–220.

Huang YH, Bergles DE (2004). Glutamate transporters bring competition to the synapse. Cur Opin Neurobiol14: 346–352.

Kanai Y, Hediger MA (2003). The glutamate and neutral amino acid transporter family: physiological and pharmacological implications. Eur J Pharmacol479: 237–247.

Kanai Y, Hediger MA (2004). The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflügers Archiv447: 465–468.

Kanner BI, Borre L (2002). The dual-function glutamate transporters: structure and molecular characterisation of the substrate-binding sites. Biochim Biophys Acta1555: 92–95.

Kanner BI (2006). Structure and function of sodium-coupled GABA and glutamate transporters. J Membr Biol213: 89–100.

Kanner BI, Zomot E (2008). Sodium-coupled neurotransmitter transporters. Chem Rev108:1654–1668.

Palacín M, Estévez R, Bertran J, Zorano A (1998). Molecular biology of mammalian plasma membrane amino acid transporters Physiol Rev78: 969–1054.

Reimer RJ, Edwards RH (2004). Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflügers Archiv447: 629–635.

Ryan RM, Vandenberg RJ (2005). A channel in a transporter. Clin Exp Pharmacol Physiol32: 1–6.

Sheldon AL, Robinson MB (2007). The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochem Int51: 333–355.

Shigeri Y, Seal RP, Shimamoto K (2004). Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Res Brain Res Rev45: 250–265.

Shimamoto K (2008). Glutamate transporter blockers for elucidation of the function of excitatory neurotransmission systems. Chem Rec8: 182–199.

Sonders MS, Quick M, Javitch JA (2005). How did the neurotransmitter cross the bilayer? A closer look. Curr Opin Neurobiol15: 296–304.

Tzingouris AV, Wadiche JI (2007). Glutamate transporters: confining runaway excitation by shaping synaptic transmission. Nat Rev Neurosci8: 935–947.

Vandenberg RJ (2006). Mutational analysis of glutamate transporters. Handb Exp Pharmacol175: 113–135.

Vandenberg RJ, Huang S, Ryan RM (2008). Slips, leaks and channels in glutamate transporters. Channels (Austin)2: 51–58.

Vandenberg RJ, Ju P, Aubrey KR, Ryan RM, Mitrovic AD (2004). Allosteric modulation of neurotransmitter transporters at excitatory synapses. Eur J Pharm Sci23: 1–11.

Vandenberg RJ, Ryan RM (2005). How and why are channels in transporters? Sci STKE2005(280): pe17.