Mammalian Excitatory Amino Acid Transporters Research Articles

Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1

Excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate from the synaptic cleft and maintain glutamate concentrations below neurotoxic levels. Recently, the crystal structures of thermostable EAAT1 variants have been reported; however, little is understood regarding the functional mechanism of the transmembrane domain (TM) 4b-4c loop, which contains more than 50 amino acids in mammalian EAATs that are absent in prokaryotic homologues. To explore the spatial position and function of TM4 during the transport cycle, we introduced pairwise cysteine substitutions between the TM4b-4c loop and TM8 in a cysteine-less version of EAAT1, CL-EAAT1. We observed pronounced inhibition of transport by Cu(II)(1,10-phenanthroline)3 (CuPh) for doubly substituted V238C/I469C and A243C/I469C variants, but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine variants. Dithiothreitol treatment partially restored the uptake activity of the CuPh-treated V238C/I469C and A243C/I469C doubly substituted variants, confirming that the effects of CuPh on these variants were due to the formation of intramolecular disulfide bonds. Glutamate, KCl, and d,l-threo-β-benzyloxy-aspartate weakened CuPh inhibition of the V238C/I469C variant, but only KCl weakened CuPh inhibition of the V243C/I469C variant, suggesting that the TM4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1. Our results suggest that the TM4b-4c loop and TM8 are positioned in close proximity during the transport cycle and are less closely spaced in the inward-facing conformation.

Substrate-Induced Motion between TM4 and TM7 of the Glutamate Transporter EAAT1 Revealed by Paired Cysteine Mutagenesis.

To maintain efficient synaptic communication, glutamate transporters reuptake glutamate from the synaptic cleft and prevent glutamate concentrations from reaching neurotoxic levels. The number of amino acid residues of the transmembrane (TM) domain 4b-4c loop of mammalian excitatory amino acid transporters (EAATs) is 50 amino acids more than that of the prokaryotic homolog. To investigate the spatial proximity and functional significance of residues in glutamate transporters, cysteine pairs were introduced at positions A243 of the TM4b-4c loop and T396 or A414 of TM7, respectively. The transport activity of double mutants A243C/T396C and A243C/A414C was inhibited by Cu(II) (1,10-phenanthroline)3 [copper phenanthroline (CuPh)] and cadmium ions, but the uptake activity of corresponding single mutants remained unchanged. Treatment with dithiothreitol after CuPh restored much of the transport activity. The inhibitory effects of CuPh and cadmium could only be detected when cysteine pairs are in the same polypeptide. Therefore, we suggest that the formation of these disulfide bonds occurs intramolecularly. Glutamate, potassium, and DL-threo-β-benzyloxyaspartate facilitated crosslinking in the A243C/T396C transporter and this suggests that the TM4b-4c loop and β-bridge region in TM7 were drawn into close proximity to each other in the inward- and outward-facing conformation of EAAT1. Thus, these data provide evidence that substrate-induced structural rearrangements occur between the TM4b-4c loop and TM7 during the transport cycle.

Allosteric Mechanisms of Molecular Machines at the Membrane: Transport by Sodium-Coupled Symporters

Solute transport across cell membranes is ubiquitous in biology as an essential physiological process. Secondary active transporters couple the unfavorable process of solute transport against its concentration gradient to the energetically favorable transport of one or several ions. The study of such transporters over several decades indicates that their function involves complex allosteric mechanisms that are progressively being revealed in atomistic detail. We focus on two well-characterized sodium-coupled symporters: the bacterial amino acid transporter LeuT, which is the prototype for the "gated pore" mechanism in the mammalian synaptic monoamine transporters, and the archaeal GltPh, which is the prototype for the "elevator" mechanism in the mammalian excitatory amino acid transporters. We present the evidence for the role of allostery in the context of a quantitative formalism that can reconcile biochemical and biophysical data and thereby connects directly to recent insights into the molecular structure and dynamics of these proteins. We demonstrate that, while the structures and mechanisms of these transporters are very different, the available data suggest a common role of specific models of allostery in their functions. We argue that such allosteric mechanisms appear essential not only for sodium-coupled symport in general but also for the function of other types of molecular machines in the membrane.

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na(+) binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na(+) requires a very high activation energy (Ea 106.8 kJ mol(-1)) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na(+) concentration present. Binding of aspartate was not observed in the absence of Na(+), whereas in the presence of high Na(+) concentrations (above the Kd for Na(+)) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 10(5) m(-1)s(-1)), with low Ea and Q10 values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na(+) binding is most likely the rate-limiting step for substrate binding.

Molecular Dynamics Simulation Study of a Mutant Construct of the Archaeal Glutamate Transporter GltPh with Transport Rates as Fast as its Human Counterpart

The glutamate transporter GltPh is a homolog of mammalian excitatory amino acid transporters (EAATs) that mediate glutamate re-uptake after discharge at the neuronal synaptic cleft, thereby enabling repeated signaling cycles and preventing excitotoxicity. While the structure of EAATs is not yet known, several stages of the transport cycle have been captured in crystallographic studies of the homotrimeric GltPh, suggesting a mechanism of separate elevator-like motions of three transport domains relative to a scaffold composed of the trimerization domains from each monomer. Dynamic aspects of the transport cycle monitored in single-molecule FRET experiments, revealed quiescent phases in which GltPh appears to be “locked” in the inward- or outward-facing states. Significantly higher transport rates, typical for human EAATs, were recently reported for a GltPh construct in which key residues were mutated to mimic the sequence of mammalian EAAT1. The mutant adopted a novel “unlocked” conformation, in which the transport domains were separated from the trimerization domains. Molecular dynamics simulations of this GltPh mutant in lipid bilayers found the unlocked conformation to be unstable if the transport/trimerization domain interface is solvated by water only, but stabilized by insertion of one or several hydrophobic moieties such as lipid tails. Analysis of the effect of the mutations on the local dynamics in several known stages of the transport cycle showed that a charged side chain introduced at the protein interface with the membrane produces membrane deformation and a destabilizing energy cost due to residual hydrophobic mismatch. Free energy perturbation calculations were used to estimate the impact of the mutation in the explored stages of the transport cycle in order to gain insights on the key structural determinants of EAAT transport efficiency.

A Na + -dependent electrogenic glutamate transporter current in voltage-clamped cells of corpora allata in the cricket Gryllus bimaculatus

Application of L-glutamate (1 mM) to corpora allata cells of the adult male cricket Gryllus bimaculatus caused a membrane depolarization of 5.9+/-0.3 mV (mean +/- SE) from a resting potential of -62.2+/-1.3 mV (n=57). The underlying mechanism for this depolarization was studied by applying the two-electrode voltage-clamp technique. Application of L-glutamate (1 mM) elicited an inward current that peaked at 8.1+/-0.7 nA (n = 73) at a holding potential of-50 mV. Both L- and D-aspartate also induced an inward current of almost the same amplitude as L-glutamate, whereas D-glutamate failed to induce an inward current. Glutamate receptor agonists, such as kainate, quisqualate, alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid, and N-methyl-D-aspartate, were ineffective in eliciting inward currents. The glutamate-induced inward current did not reverse even when the holding potential was set to +40 mV. The replacement of extracellular Na+ with choline+ eliminated the inward current. These results strongly suggest that the current induced by glutamate is mediated by a glutamate transporter rather than a glutamate receptor. We further examined the effects of 12 amino acid analogs which are known to be selective inhibitors of the mammalian excitatory amino acid transporters (EAATs) on the corpora allata transporter. From the effects of these inhibitors, we conclude that the glutamate transporter expressed in corpora allata cells of the cricket is similar to the high affinity glutamate transporters cloned from human brain, especially EAAT1 and EAAT3. Unlike mammalian transporters, however, serine-O-sulfate has the most potent action, suggesting the unique feature of the glutamate transporter expressed in the corpora allata.