Induced fit substrate binding to an archeal glutamate transporter homologue

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. Glt_Ph, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) Glt_Ph in detergent micelles. At low millimolar [Na⁺], the addition of l-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na⁺], the kinetics were dominated by a fast component [k_obs,fast; K_D (Na⁺) = 22 ± 3 mM, n_Hill = 1.7 ± 0.3] with values of k_obs,fast rising in a saturable manner to ≈500 s⁻¹ (at 6 °C) with increasing [l-aspartate]. The binding kinetics of l-aspartate differed from the binding kinetics of two alternative substrates: l-cysteine sulfinic acid and d-aspartate. l-cysteine sulfinic acid bound with higher affinity than l-aspartate but involved lower saturating rates, whereas the saturating rates after d-aspartate binding were higher. Thus, after the association of two Na⁺ to the empty transporter, Glt_Ph binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for Glt_Ph than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.     

Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber-Purkinje cell synapse

Excitatory amino acid transporters (EAATs) located on neurons and glia are responsible for limiting extracellular glutamate concentrations, but specific contributions made by neuronal and glial EAATs have not been determined. At climbing fiber to Purkinje cell (PC) synapses in cerebellum, a fraction of released glutamate is rapidly bound and inactivated by neuronal EAATs located on postsynaptic PCs. Because transport involves a stoichiometric movement of ions and is electrogenic, postsynaptic currents mediated by EAATs should permit precise calculation of the amount of postsynaptic glutamate uptake. However, this is possible only if a stoichiometric EAAT current can be isolated from all other contaminating signals. We used synaptic stimulation and photolysis of caged glutamate to characterize the current in PCs that is resistant to high concentrations of glutamate receptor antagonists. Some of this response is inhibited by the high-affinity EAAT antagonist TBOA (dl-threo-beta-benzyloxyaspartic acid), whereas the remaining current shows properties inconsistent with glutamate transport. By subtracting this residual non-EAAT current from the response recorded in glutamate receptor antagonists, we have obtained an estimate of postsynaptic uptake near physiological temperature. Analysis of such synaptic EAAT currents suggests that, on average, postsynaptic EAATs take up approximately 1,300,000 glutamate molecules in response to a single climbing fiber action potential.     

A dynamic switch between inhibitory and excitatory currents in a neuronal glutamate transporter

Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission and maintain extracellular glutamate concentrations in the central nervous system below excitotoxic levels. In addition to sustaining a secondary-active glutamate transport, EAAT glutamate transporters also function as anion-selective channels. Here, we report a gating process that makes anion channels associated with a neuronal glutamate transporter, EAAT4, permeable to cations and causes a selective increase of the open probability at voltages negative to the actual current reversal potential. The activation process depends on both membrane potential and extracellular glutamate concentration and causes an accumulation of EAAT4 anion channels in a state favoring cation influx and anion efflux. Gating of EAAT4 anion channels thus allows a switch between inhibitory currents in resting cells and excitatory currents in electrically active cells. This transporter-mediated conductance could modify the excitability of Purkinje neurons, providing them with an unprecedented mechanism for adaptation.     

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Glutamate transporters are membrane proteins found in neurons and glial cells, which play a critical role in regulating cell signaling by clearing glutamate released from synapses. Although extensive biochemical and structural studies have shed light onto different aspects of glutamate transport, the time-resolved molecular mechanism of substrate (glutamate or aspartate) translocation, that is, the sequence of events occurring at the atomic level after substrate binding and before its release intracellularly remain to be elucidated. We identify an energetically preferred permeation pathway of ≈23 Å between the helix HP1b on the hairpin HP1 and the transmembrane helices TM7 and TM8, using the high resolution structure of the transporter from Pyrococcus horikoshii (Glt_Ph) in steered molecular dynamics simulations. Detailed potential of mean force calculations along the putative pathway reveal 2 energy barriers encountered by the substrate (aspartate) before it reaches the exit. The first barrier is surmounted with the assistance of 2 conserved residues (S278 and N401) and a sodium ion (Na2); and the second, by the electrostatic interactions with D405 and another sodium ion (Na1). The observed critical interactions and mediating role of conserved residues in the core domain, the accompanying conformational changes (in both substrate and transporter) that relieve local strains, and the unique coupling of aspartate transport to Na⁺ dislocation provide insights into methods for modulating substrate transport.Sodium-dependent glutamate transporters, also known as excitatory amino acid transporters (EAATs), are membrane proteins involved in regulating excitatory signal transmission by clearing excess l-glutamate released at synapses (1). The concentration of these excitatory amino acid neurotransmitters at the extracellular (EC) space may increase by several thousandfold during the periods of synaptic activation (2–4). Efficient removal of excess glutamate is critical to protect neurons against excitotoxicity. Glutamate uptake in the brain is coupled to the cotransport of 3 sodium ions (Na⁺) and 1 proton (H⁺), and followed by the countertransport of 1 potassium ion (K⁺) (2–4). In addition, in the transporter, glutamate activates an uncoupled chloride (Cl⁻) current, which is known to be important for glutamatergic neurotransmission (5–7).The first crystal structure of an archaeal orthologue (Glt_Ph, from Pyrococcus horikoshii) of the eukaryotic EAATs was resolved by Gouaux and coworkers (8) in 2004. This structure offered the possibility of exploring the molecular interactions that drive substrate transport via structure-based models and simulations. Additional insights were provided in a more recent article by Boudker et al. (9), which reported another conformation of Glt_Ph, resolved in the presence of the bound substrate and thallium ions. Radio labeled flux experiments revealed that Glt_Ph preferentially mediates the uptake of aspartate over glutamate (9).These X-ray crystallographic structures showed that Glt_Ph is a bowl-shaped homotrimer, with a solvent-filled basin exposed to the EC region (Fig. 1A). The basin extends almost halfway through the membrane bilayer. Each monomer consists of 8 transmembrane helices (TM1-TM8) and 2 helical hairpins (HP1 and HP2), organized into 2 domains (Fig. 1B). The C-terminal “core domain” facing the central basin is composed of the structural elements HP1, TM7, HP2, and TM8; and the N-terminal domain is composed of the helices TM1-TM6 on the outer part of the membrane protein. The core domain is essential in mediating Na⁺-dependent substrate transport. It is surrounded and secured within the TM helices of the N-terminal domain.Open in a separate windowFig. 1.Schematic view of Glt_Ph and surrounding lipid and water molecules. (A) Side view of the simulation box. Glt_Ph is a bowl-shaped homotrimer. Its 3 monomers are colored green, cyan, and magenta. It is embedded in the lipid bilayer (gray) and water (red) molecules. Aspartates are shown as yellow spheres in (A). (B) Close view of a monomer. Noncore residues are rendered translucent. The core domain (TM7, TM8, HP1, and HP2) is colored in rainbow scheme. The aspartate is displayed in stick representation and the 2 sodium ions as blue spheres. The structural coordinates are taken from the PDB entry 2NWX.One of the most conserved regions of the sequence is the N₃₁₀MDGT₃₁₄ motif at the partially unwound portion of TM7, between the 2 helical segments TM7a and TM7b. This motif has been pointed out to play a role in binding the substrate and sodium ions, as also confirmed by the Glt_Ph structure (4, 8). In addition, residues in the mammalian EAATs equivalent to the Glt_Ph residues D394 and R397 on TM8 and the serine rich motif (S277-S278-S279) at the HP1 tip have been proposed to be involved in glutamate binding, in line with the interactions with the bound aspartate observed in the Glt_Ph structure (8–10).We recently examined the substrate recognition and binding events of Glt_Ph (11) by molecular dynamic (MD) simulations in a fully solvated lipid-bilayer. The helical hairpin HP2 was observed to open up and close down multiple times during 40 ns runs performed in the absence of substrate. These simulations thus confirmed that the HP2 hairpin acts as an “EC gate” that readily fluctuates between open and closed states. Moreover, they showed that the open state is predisposed for substrate binding within nanoseconds, and the closed state is favored upon Na⁺ binding. No net correlation was observed between the motions of the 3 EC gates, confirming that the subunits function independently with respect to their own substrate neighborhoods. However, no further permeation or release to the cell interior could be observed. The translocation of substrate has indeed a timescale of milliseconds, beyond the reach of conventional MD.In the present study, we focus on the translocation of the substrate from its binding site near the EC gate into the transporter interior and its release to the cytoplasm. Our objective is to elucidate the translocation pathway(s) and corresponding free energy profile, and understand the significance of Na⁺ cotransport. To this aim, we conducted a series of steered MD simulations (12) using Glt_Ph structure and its substrate, aspartate. Steered MD permits us to accelerate the translocation process and thereby identify the pathway(s) preferentially selected by the substrate, and the coupling to Na⁺ cotransport. A highly preferred pathway, called P1, is revealed between the helices HP1b (of hairpin HP1), TM7, and TM8, along with an alternative, less probable outlet (P2). The Na⁺-dependent stochastic events that facilitate the channeling of the substrate to the exit point are visualized at the atomic level detail. We calculated the potential of mean force (PMF) profile using umbrella sampling and a weighted histogram analysis method (WHAM) (13, 14) applied to a total of 46 windows along the 23-Å-long permeation pathway. Two local energy barriers were identified. The conformational changes coupled to Na⁺ dislocation undergone by the substrate and surrounding transporter residues for relieving the local strains near the energy barriers provide us with insights into the molecular mechanism and atomic interactions that control substrate transport by this important family of transporters.     

The role of excitatory amino acid transporter 2 (EAAT2) in epilepsy and other neurological disorders

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). Excitatory amino acid transporters (EAATs) have important roles in the uptake of glutamate and termination of glutamatergic transmission. Up to now, five EAAT isoforms (EAAT1-5) have been identified in mammals. The main focus of this review is EAAT2. This protein has an important role in the pathoetiology of epilepsy. De novo dominant mutations, as well as inherited recessive mutation in this gene, have been associated with epilepsy. Moreover, dysregulation of this protein is implicated in a range of neurological diseases, namely amyotrophic lateral sclerosis, alzheimer’s disease, parkinson’s disease, schizophrenia, epilepsy, and autism. In this review, we summarize the role of EAAT2 in epilepsy and other neurological disorders, then provide an overview of the therapeutic modulation of this protein.

     

The 3-4 loop of an archaeal glutamate transporter homolog experiences ligand-induced structural changes and is essential for transport

Glutamatergic synaptic transmission is terminated by members of the excitatory amino acid transporter (EAAT) family of proteins that remove glutamate from the synaptic cleft by transporting it into surrounding glial cells. Recent structures of a bacterial homolog suggest that major motions within the transmembrane domain translocate the substrate across the membrane. However, the events leading to this large structural rearrangement are much less clear. Two reentrant loops have been proposed to act as extracellular and intracellular gates, but whether other regions of these proteins play a role in the transport process is unknown. We hypothesized that transport-related conformational changes could change the solvent accessibilities of affected residues, as reflected in protease sensitivity or small-molecule reactivity. In the model system Glt_Ph, an archaeal EAAT homologue from Pyrococcus horikoshii, limited trypsin proteolysis experiments initially identified a site in the long extracellular loop that stretches between helices 3 and 4 that becomes protected from proteolysis in the presence of a substrate, L-aspartate, or an inhibitor, DL-TBOA in the presence of Na⁺, the cotransported ion. Using a combination of site-directed cysteine-scanning mutagenesis and fluorescein-5-maleimide labeling we found that positions throughout the loop experience these ligand-induced conformational changes. By selectively cleaving the 3-4 loop (via introduced Factor Xa sites) we demonstrate that it plays a vital role in the transport process; though structurally intact, the cleaved proteins are unable to transport aspartate. These results inculcate the 3-4 loop as an important player in the transport process, a finding not predicted by any of the available crystal structures of Glt_Ph.     

Ethanol increases the activity of rat excitatory amino acid transporter type 4 expressed in Xenopus oocytes: role of protein kinase C and phosphatidylinositol 3-kinase

Background: Glutamate is the major excitatory neurotransmitter in the central nervous system and is critical for essentially all physiological processes, such as learning, memory, central pain transduction, and control of motor function. Excitatory amino acid transporters (EAATs) play a key role in regulating glutamate neurotransmission by uptake of glutamate into cells. EAAT4 is the major EAAT in the cerebellar Purkinje cells. The authors investigated the effects of ethanol on EAAT4 and the mediatory effects of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in this context. Methods: Excitatory amino acid transporter 4 was expressed in Xenopus oocytes by injecting EAAT4 mRNA. l ‐aspartate‐induced membrane currents were measured using a two‐electrode voltage clamp. Responses were quantified by integrating current traces and are represented in microCoulombs (μC). Results: Ethanol increased EAAT4 activity in a dose‐dependent manner. At ethanol concentrations of 25, 50, 100, and 200 mM, the responses were significantly higher than untreated control values. Ethanol (25 mM) significantly increased the V_max (1.5 ± 0.1 μC for control vs. 2.0 ± 0.1 μC for ethanol, p < 0.05), but did not affect K_m (2.3 ± 0.6 μM for control vs. 1.7 ± 0.7 μM for ethanol, p > 0.05) of EAAT4 for l ‐aspartate. Preincubation of oocytes with phorbol‐12‐myristate‐13‐acetate (PMA, a PKC activator) significantly increased EAAT4 activity. However, combinations of PMA and ethanol versus PMA or ethanol alone did not increase responses further. Two PKC inhibitors, chelerythrine and staurosporine did not reduce basal EAAT4 activity but abolished ethanol‐enhanced EAAT4 activity. Pretreatment with wortmannin (a PI3K inhibitor) also abolished ethanol‐enhanced EAAT4 activity. Conclusions: These results demonstrate that acute ethanol exposure increases EAAT4 activity at clinically relevant concentrations and that PKC and PI3K may mediate this. The effects of ethanol on EAAT4 may play a role in the cerebellar dysfunction caused by ethanol intoxication.     

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K_m and K_d for sodium, as measured by betaine uptake, tryptophan fluorescence, and ²²Na⁺ binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.     

Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance.

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.     

The equivalent of a thallium binding residue from an archeal homolog controls cation interactions in brain glutamate transporters

Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. Their transport cycle consists of two separate translocation steps, namely cotransport of glutamic acid with three Na⁺ followed by countertransport of K⁺. Two Tl⁺ binding sites, presumed to serve as sodium sites, were observed in the crystal structure of a related archeal homolog and the side chain of a conserved aspartate residue contributed to one of these sites. We have mutated the corresponding residue of the eukaryotic glutamate transporters GLT-1 and EAAC1 to asparagine, serine, and cysteine. Remarkably, these mutants exhibited significant sodium-dependent radioactive acidic amino acid uptake when expressed in HeLa cells. Reconstitution experiments revealed that net uptake by the mutants in K⁺-loaded liposomes was impaired. However, with Na⁺ and unlabeled L-aspartate inside the liposomes, exchange levels were around 50–90% of those by wild-type. In further contrast to wild-type, where either substrate or K⁺ stimulated the anion conductance by the transporter, substrate but not K⁺ modulated the anion conductance of the mutants expressed in oocytes. Both with wild-type EAAC1 and EAAC1-D455N, not only sodium but also lithium could support radioactive acidic amino acid uptake. In contrast, with D455S and D455C, radioactive uptake was only observed in the presence of sodium. Thus the conserved aspartate is required for transporter-cation interactions in each of the two separate translocation steps and likely participates in an overlapping sodium and potassium binding site.     

Intrinsic kinetics determine the time course of neuronal synaptic transporter currents

Efficient clearance of synaptically released glutamate from the extracellular space is an absolute requirement for maintaining information processing in the central nervous system. In the cerebellum, clearance of glutamate relies on uptake by Bergmann glial cells and Purkinje cells (PCs). Uptake by PCs can be monitored by recording the synaptic transport current (STC) mediated by the PC-specific transporter excitatory amino acid transporter 4 (EAAT4). The slow time course of the PC STC has been used to argue that glutamate clearance is protracted. We find, however, that the time course of the STC is not affected by altering the amount of glutamate released at individual synapses or by partial transporter blockade, manipulations that would be expected to change the duration of the extracellular glutamate transient. Ion substitution experiments and kinetic modeling of the PC transporter current suggest that physiological levels of intracellular Na(+) and glutamate slow the cycling rate of transporters and thereby lengthen the time course of STCs. The model predicts that PC transporters bind glutamate quickly but that the actual cycling rate of EAAT4 in physiological conditions is slow; therefore, the STC reflects the intrinsic kinetics of the glutamate transporter, not the rate of glutamate clearance.     

ENT1 Regulates Ethanol‐Sensitive EAAT2 Expression and Function in Astrocytes

Background: Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum. Methods: We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real‐time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol‐induced EAAT2 expression. Results: An ENT1‐specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up‐regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol‐induced EAAT2 up‐regulation. Conclusions: Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.     

新生大鼠缺血缺氧后胶质性谷氨酸转运体的表达及GM1的干预研究

目的探讨新生大鼠缺血缺氧性脑损伤后胶质性谷氨酸转运体的表达及神经节苷脂（GM1）的干预作用。方法通过建立新生大鼠缺血缺氧性脑损伤动物模型，应用免疫组化方法，观察缺血缺氧后不同时期大脑皮质胶质性谷氨酸转运体EAATI、EAAT2的动态表达度GM1对其表达的影响。结果缺血缺氧后6hEAAT1的表达开始上升、第2d达高峰，第3d恢复到假手术组水平；EAAT2的表达在缺血缺氧后12h开始上升，第3d达高峰，第5d恢复到假手术组水平；GM1干预组脑组织损伤明显减轻，EAAT1和EAAT2的表达较单纯缺血缺氧组显著增加（P〈0．01），持续时间延长。结论缺血缺氧诱导胶质性谷氨酸转运体的表达，GM1提高胶质性谷氨酸转运体的表达可能是GM1脑保护作用的重要机制之一。     

Glutamate uptake in retinal glial cells during diabetes

Aims Glutamate recycling is a major function of retinal Müller cells. The aim of this study was to evaluate the expression and function of glutamate transporters during diabetes.Methods Sprague–Dawley rats were rendered diabetic by a single dose of streptozotocin (50 mg/kg). Following 12 weeks of diabetes, immunolocalisation and mRNA expression of the two glial cell transporters, GLAST and EAAT4 were evaluated using indirect immunofluorescence and real-time PCR. The function of glutamate transport was investigated at 1, 4 and 12 weeks following induction of diabetes by measuring the level of uptake of the non-metabolisable glutamate analogue, d-aspartate, into Müller cells.Results There was no difference in the localisation of either GLAST or EAAT4 during diabetes. Although there was a small apparent increase in expression of both GLAST and EAAT4 in diabetic retinae compared with controls this was not statistically significant. At 1, 4 and 12 weeks following diabetes, d-aspartate immunoreactivity was significantly increased in Müller cells of diabetic rats compared to controls (p<0.001). The EC₅₀ was found to increase by 0.304 log units in diabetic Müller cells compared with controls, suggesting that glutamate uptake is twice as efficient.Conclusions These data suggest that there are alterations in glutamate transport during diabetes. However, these changes are unlikely to play a significant role in glutamate-induced neuronal excitoxicity during diabetes. These results suggest that although Müller cells undergo gliosis at an early stage of diabetes, one of the most important functions for maintaining normal retinal function is preserved within the retina.     

Two serine residues of the glutamate transporter GLT-1 are crucial for coupling the fluxes of sodium and the neurotransmitter

The neurotoxicity of glutamate in the central nervous system is restricted by several (Na⁺ + K⁺)-coupled transporters for this neurotransmitter. The astroglial transporter GLT-1 is the only subtype that exhibits high sensitivity to the nontransportable glutamate analogue dihydrokainate. A marked reduction in sensitivity to the blocker is observed when serine residues 440 and 443 are mutated to glycine and glutamine, which, respectively, occupy these positions in the other homologous glutamate transporters. They are located in the ascending limb of the recently identified pore-loop-like structure. Strikingly, mutation of serine-440 to glycine enables not only sodium but also lithium ions to drive net influx of acidic amino acids. Moreover, the efficiency of lithium as a driving ion for glutamate transport depends on the nature of the amino acid residue present at position 443. Mutant transporters containing single cysteines at the position of either serine residue become sensitive to positively as well as negatively charged methanethiosulfonate derivatives. In S440C transporters significant protection against this inhibition is provided both by transportable and nontransportable glutamate analogues, but not by sodium alone. Our observations indicate that the pore-loop-like structure plays a pivotal role in coupling ion and glutamate fluxes and suggest that it is close to the glutamate-binding site.     

Inhibition of the activity of excitatory amino acid transporter 4 expressed in Xenopus oocytes after chronic exposure to ethanol

Background: The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in these effects. Methods: EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol‐containing solution for 24 to 96 hours. Membrane currents induced by l ‐aspartate were recorded using 2‐electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (μC). Results: Ethanol dose‐ and time‐dependently reduced EAAT4 activity. EAAT4 activity after a 96‐hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 μC for control vs. 1.6 ± 0.2 μC for ethanol, n = 18, p < 0.05) of EAAT4 for l ‐aspartate. Preincubation of ethanol‐treated (50 mM for 96 hours) oocytes with phorbol‐12‐myrisate‐13‐acetate (100 nM for 10 minutes) abolished the ethanol‐induced decrease in EAAT4 activity. While staurosporine (2 μM for 1 hour) or chelerythrine (100 μM for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 μM for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions: The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri‐Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion.     

A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter

Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O₂ to H₂O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled (“gating” region) overlaps with a channel through which O₂ is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O₂ access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O₂ delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.     

The central cavity in trimeric glutamate transporters restricts ligand diffusion

A prominent aqueous cavity is formed by the junction of three identical subunits in the excitatory amino acid transporter (EAAT) family. To investigate the effect of this structure on the interaction of ligands with the transporter, we recorded currents in voltage-clamped Xenopus oocytes expressing EAATs and used concentration jumps to measure binding and unbinding rates of a high-affinity aspartate analog that competitively blocks transport (β-2-fluorenyl-aspartylamide; 2-FAA). The binding rates of the blocker were approximately one order of magnitude slower than l-Glu and were not significantly different for EAAT1, EAAT2, or EAAT3, but 2-FAA exhibited higher affinity for the neuronal transporter EAAT3 as a result of a slower dissociation rate. Unexpectedly, the rate of recovery from block was increased by l-Glu in a saturable and concentration-dependent manner, ruling out a first-order mechanism and suggesting that following unbinding, there is a significant probability of ligand rebinding to the same or neighboring subunits within a trimer. Consistent with such a mechanism, coexpression of wild-type subunits with mutant (R447C) subunits that do not bind glutamate or 2-FAA also increased the unblocking rate. The data suggest that electrostatic and steric factors result in an effective dissociation rate that is approximately sevenfold slower than the microscopic subunit unbinding rate. The quaternary structure, which has been conserved through evolution, is expected to increase the transporters' capture efficiency by increasing the probability that following unbinding, a ligand will rebind as opposed to being lost to diffusion.