Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1

Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure–function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)₃ (CuPh) and cadmium ion (Cd²⁺) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + l-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.     

A complex relative motion between hairpin loop 2 and transmembrane domain 5 in the glutamate transporter GLT-1

Excitatory amino acid transporter 2, also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining suitable synaptic glutamate concentrations. Reentrant helical hairpin loop (HP) 2, as the extracellular gate, has been shown to participate in the binding of substrate and ions. Several residues in transmembrane domain (TM) 5 have been shown to be involved in the construction of the transport pathway. However, the spatial relationship between HP2 and TM5 during the recycling of glutamate has not yet been clarified. We introduced cysteine residue pairs in HP2 and TM5 of cysteine-less-GLT-1 by using site-directed mutagenesis in order to assess the proximity of HP2 and TM5. A significant decrease in substrate uptake was seen in the I283C/S443C and S287C/S443C mutants when the oxidative cross-linking agent copper(II) (1,10-phenanthroline)₃ (CuPh) was used. The inhibitory effect of CuPh on the transport activity of the S287/S443C mutant was increased after the application of glutamate or potassium. In contrast, an apparent protection of the transport activity of the I283C/S443C mutant was observed after glutamate or potassium addition. The membrane-impermeable sulfhydryl reagent (2-trimethylammonium) methanethiosulfonate (MTSET) was used to detect the aqueous permeability of each single mutant. The aqueous permeability of the I283C mutant was identical to that of the S443C mutant. The sensitivity of I283C and S443C to MTSET was attenuated by glutamate and potassium. All these data indicate that there is a complex relative motion between TM5 and HP2 during the transport cycle.     

Substrates and non-transportable analogues induce structural rearrangements at the extracellular entrance of the glial glutamate transporter GLT-1/EAAT2

To explore rearrangements of the reentrant loop HP2 relative to transmembrane domains (TMs) 7 and 8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. The pairs were introduced around 10-15 A "above" the residues, which make contact with substrate in the related archaeal homologue Glt(Ph). Transport by the double mutants M449C/L466C (HP2/TM 8), L453C/I463C (HP2/TM 8), and I411C/I463C (TM 7/TM 8) was inhibited by copper(II)(1,10-phenanthroline)(3) (CuPh) and by Cd(2+). Inhibition was only observed when the two cysteines were present in the same construct, but not with the respective single cysteine mutants or when only one cysteine was paired with a mutation to another residue. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of M449C/L466C by CuPh. The non-transportable analogues kainate and d, l-threo-beta-benzyloxyaspartate are expected to stabilize an outward-facing conformation, but only the latter potentiated the effect of CuPh on M449C/L466C. However, both analogues increased the aqueous accessibility of the cysteines introduced at positions 449 and 466 to a membrane-impermeant sulfhydryl reagent. Inhibition of L453C/I463C by CuPh was protected not only by glutamate but also by the two analogues. In contrast, these ligands had very little effect on the inhibition of I411C/I463C by CuPh. Our results are consistent with the proposal that HP2 serves as the extracellular gate of the transporter and indicate that glutamate and the two analogues induce distinct conformations of HP2.     

The aqueous accessibility in the external half of transmembrane domain I of the GABA transporter GAT-1 Is modulated by its ligands

The sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transporter GAT-1 is the first identified member of a family of transporters, which maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. To obtain evidence for the idea that the highly conserved transmembrane domain I (TMD I) participates in the permeation pathway, we have determined the impact of impermeant methanethiosulfonate (MTS) reagents on cysteine residues engineered into this domain. As a background the essentially insensitive but fully active C74A mutant has been used. Transport activity of mutants with a cysteine introduced cytoplasmic to glycine 63 is largely unaffected and is resistant to the impermeant MTS reagents. Conversely, transport activity in mutants extracellular to glycine 63 is strongly impacted. Nevertheless, transport activity could be measured in all but three mutants: G65C, N66C, and R69C. In each of the six active cysteine mutants the activity is highly sensitive to the impermeant MTS reagents. This sensitivity is potentiated by sodium in L64C, F70C, and Y72C, but is protected in V67C and P71C. GABA protects in L64C, W68C, F70C, and P71C. The non-transportable GABA analogue SKF100330A also protects in L64C, W68C, and P71C as well as V67C, but strikingly potentiates inhibition in F70C. Although cysteine substitution in this region may have perturbed the native structure of GAT-1, our observations, taken together with the recently published accessibility study on the related serotonin transporter (Henry, L. K., Adkins, E. M., Han, Q., and Blakely, R. D. (2003) J. Biol. Chem. 278, 37052-37063), suggest that the extracellular part of TMD I is conformationally sensitive, lines the permeation pathway, and forms a more extended structure than expected from a membrane-embedded alpha-helix.     

The accessibility in the external part of the TM5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle

Background

Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM) 5 of the EAAT1 are not clear yet.

Methodology/Principal Findings

We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. V_max of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA.

Conclusions/Significance

All these facts suggest that the accessibility of some positions of the external part of the TM5 is conformationally sensitive during the transport cycle. Our results indicate that some residues of TM5 take part in the transport pathway during the transport cycle.     

An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1

GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.     

Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis

Background

GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt_Ph, it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)₃ (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1.

Methodology/Principal Findings

To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)₃ is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd²⁺.

Conclusions/Significance

Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle.     

The reactivity of the gamma-aminobutyric acid transporter GAT-1 toward sulfhydryl reagents is conformationally sensitive. Identification of a major target residue.

The gamma-aminobutyric acid (GABA) transporter GAT-1 is a prototype of neurotransmitter transporters that maintain low synaptic levels of the transmitter. Transport by GAT-1 is sensitive to the polar sulfhydryl reagent 2-aminoethyl methanethiosulfonate. Following replacement of endogenous cysteines to other residues by site-directed mutagenesis, we have identified cysteine 399 as the major determinant of the sensitivity of the transporter to sulfhydryl modification. Cysteine-399 is located in the intracellular loop connecting putative transmembrane domains eight and nine. Binding of both sodium and chloride leads to a reduced sensitivity to sulfhydryl reagents, whereas subsequent binding of GABA increases it. Strikingly binding of the nontransportable GABA analogue SKF100330A gives rise to a marked protection against sulfhydryl modification. These effects were not observed in C399S transporters. Under standard conditions GAT-1 is almost insensitive toward the impermeant 2-(trimethylammonium)ethyl methanethiosulfonate. However, in a chloride-free medium, addition of SKF100330A renders wild type GAT-1, but not C399S, very sensitive to this impermeant reagent. These observations indicate that the accessibility of cysteine 399 is highly dependent on the conformation of GAT-1. Consequently, topological assignments based on accessibility of endogeneous or engineered cysteines to small polar sulfhydryl reagents need to be interpreted with extreme caution.     

Serotonin and cocaine-sensitive inactivation of human serotonin transporters by methanethiosulfonates targeted to transmembrane domain I

To explore aqueous accessibility and functional contributions of transmembrane domain (TM) 1 in human serotonin transporter (hSERT) proteins, we utilized the largely methanethiosulfonate (MTS) insensitive hSERT C109A mutant and mutated individual residues of hSERT TM1 to Cys followed by tests of MTS inactivation of 5-hydroxytryptamine (5-HT) transport. Residues in TM1 cytoplasmic to Gly-94 were largely unaffected by Cys substitution, whereas the mutation of residues extracellular to Ile-93 variably diminished transport activity. TM1 Cys substitutions displayed differential sensitivity to MTS reagents, with residues more cytoplasmic to Asp-98 being largely insensitive to MTS inactivation. Aminoethylmethanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) similarly and profoundly inactivated 5-HT transport by SERT mutants D98C, G100C, W103C, and Y107C. MTSEA uniquely inactivated transport activity of S91C, G94C, Y95C but increased activity at I108C. MTSEA and MTSET, but not MTSES, inactivated transport function at N101C. Notably, 5-HT provided partial to complete protection from MTSET inactivation for D98C, G100C, N101C, and Y107C. Equivalent blockade of MTSET inactivation at N101C was observed with 5-HT at both room temperature and at 4 degrees C, inconsistent with major conformational changes leading to protection. Notably, cocaine also protected MTSET inactivation of G100C and N101C, although MTS incubations with N101C that eliminate 5-HT transport do not preclude cocaine analog binding nor its inhibition by 5-HT. 5-HT modestly enhanced the inactivation by MTSET at I93C and Y95C, whereas cocaine significantly enhanced MTSET sensitivity at Y107C and I108C. In summary, our studies reveal physical differences in TM1 accessibility to externally applied MTS reagents and reveal sites supporting substrate and antagonist modulation of MTS inactivation. Moreover, we identify a limit to accessibility for membrane-impermeant MTS reagents that may reflect aspects of an occluded permeation pathway.     

Evidence that the TM1-TM2 loop contributes to the rho1 GABA receptor pore

Considerable evidence indicates the second transmembrane domain (TM2) of the gamma-aminobutyric acid (GABA) receptor lines the integral ion pore. To further delineate the structures that constitute the ion pore and selectivity filter of the rho1 GABA receptor, we used the substituted cysteine accessibility method with charged reagents to identify anion- and cation-accessible surfaces. Twenty-one consecutive residues were mutated to cysteine, one at a time, in the presumed intracellular end of the first transmembrane domain (TM1; Ala(271)-Met(276)), the entire linker connecting TM1 to TM2 (Leu(277)-Arg(287)), and the presumed intracellular end of TM2 (Ala(288)-Ala(291)). Positively (MTSEA(+)) and negatively (pCMBS(-)) charged sulfhydryl reagents, as well as Cd(2+), were added extracellularly to test accessibility of the engineered cysteines. Four of the mutants, all at the intracellular end of TM2 (R287C, V289C, P290C, A291C), were accessible to positively charged reagents, whereas seven mutants (A271C, T272C, L277C, W279C, V280C, P290C, A291C) were functionally modified by negatively charged pCMBS(-). These seven modified residues were at the intracellular end of TM2, in the TM1-TM2 linker, and at the intracellular end of TM1. In nearly all cases (excluding P290C), the rate and the degree of modification were state-dependent, with greater accessibility in the presence of agonist. Select cysteine mutants were combined with a point mutation (A291E) that converted the pore from chloride- to non-selective. In this case, positively charged reagents could modify residues in the TM1-TM2 linker (Leu(277) and Val(280)), supporting the notion that the modifying reagents were reaching their target through the pore. Taken together, our results suggest that, up to its intracellular end, the TM2 domain is not charge selective. In addition, we propose that the TM1-TM2 linker and the intracellular end of TM1 are along the pathway of the permeating ion. These findings may lend new insights into the structure of the GABA receptor pore.     

Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1

The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.     

Functional Consequences of Sulfhydryl Modification of the γ-Aminobutyric Acid Transporter 1 at a Single Solvent-Exposed Cysteine Residue

The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of ??-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1?C2.5?mM for 5?C20?min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.     

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes

The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.     

Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1

GABA transporters play an important but poorly understood role in neuronal inhibition. They can reverse, but this is widely thought to occur only under pathological conditions. Here we use a heterologous expression system to show that the reversal potential of GAT-1 under physiologically relevant conditions is near the normal resting potential of neurons and that reversal can occur rapidly enough to release GABA during simulated action potentials. We then use paired recordings from cultured hippocampal neurons and show that GABAergic transmission is not prevented by four methods widely used to block vesicular release. This nonvesicular neurotransmission was potently blocked by GAT-1 antagonists and was enhanced by agents that increase cytosolic [GABA] or [Na(+)] (which would increase GAT-1 reversal). We conclude that GAT-1 regulates tonic inhibition by clamping ambient [GABA] at a level high enough to activate high-affinity GABA(A) receptors and that transporter-mediated GABA release can contribute to phasic inhibition.     

Conformationally Sensitive Proximity of Extracellular Loops 2 and 4 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis

The sodium- and chloride-coupled GABA transporter GAT-1 is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Structural work on the bacterial homologue LeuT suggests that extracellular loop 4 closes the extracellular solvent pathway when the transporter becomes inward-facing. To test whether this model can be extrapolated to GAT-1, cysteine residues were introduced at positions 359 and 448 of extracellular loop 4 and transmembrane helix 10, respectively. Treatment of HeLa cells, expressing the double cysteine mutant S359C/K448C with the oxidizing reagent copper(II)(1,10-phenantroline)₃, resulted in a significant inhibition of [³H]GABA transport. However, transport by the single cysteine mutant S359C was also inhibited by the oxidant, whereas its activity was almost 4-fold stimulated by dithiothreitol. Both effects were attenuated when the conserved cysteine residues, Cys-164 and/or Cys-173, were replaced by serine. These cysteines are located in extracellular loop 2, the role of which in the structure and function of the eukaryotic neurotransmitter:sodium:symporters remains unknown. The inhibition of transport of S359C by the oxidant was markedly reduced under conditions expected to increase the proportion of inward-facing transporters, whereas the reactivity of the mutants to a membrane-impermeant sulfhydryl reagent was not conformationally sensitive. Our data suggest that extracellular loops 2 and 4 come into close proximity to each other in the outward-facing conformation of GAT-1.     

GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated inhibition in the cerebral cortex

γ-Aminobutyric acid 1 (GAT-1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT-1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT-2 and GAT-3 levels in KOs were unchanged and that GAT-3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl-evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABA_AR-mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post-synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABA_AR-mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT-1 KO mice GAT-3 is not redistributed and GADs are adaptively changed and indicate that GAT-1 has a prominent role in both tonic and phasic GABA_AR-mediated inhibition, in particular during sustained neuronal activity.     

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K_m. We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an α-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60° arc suggesting that TM11 is largely α helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.     

Different residues in the GABA(A) receptor alpha 1T60-alpha 1K70 region mediate GABA and SR-95531 actions

Although gamma-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from alpha(1)T60 to alpha(1)K70 was mutated to cysteine and expressed with wild-type beta(2) subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with alpha(1)T60C, alpha(1)D62C, alpha(1)F64C, alpha(1)R66C, alpha(1)S68C, and alpha(1)K70C. gamma-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of alpha(1)F64C, alpha(1)R66C, and alpha(1)S68C, whereas SR-95531 slowed modification of alpha(1)D62C, alpha(1)F64C, and alpha(1)R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for alpha(1)F64C and alpha(1)R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized alpha(1)F64C and alpha(1)R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of alpha(1)D62C and alpha(1)S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.     

The interaction of the gamma-aminobutyric acid transporter GAT-1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive cysteine residue engineered into extracellular loop IV

The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by cysteine scanning mutagenesis. Fourteen residues of this loop have been replaced by cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible cysteine residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.