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Glial Glutamate Transporters: Electrophysiology
Glial Glutamate Transporters: Electrophysiology
805
Glial Glutamate Transporters: Electrophysiology M P Kavanaugh, University of Montana, Missoula, MT, USA ã 2009 Elsevier Ltd. All rights reserved.
Molecular Diversity and Expression Patterns The mammalian glutamate transporter gene family has five excitatory amino acid transporter (EAAT) members (EAAT1–EAAT5) that are expressed in the brain in highly specific regional and cell-type patterns (see Table 1). In some cases, transporter transcripts undergo alternative splicing, adding further complexity to these expression patterns. EAAT cDNAs have predicted amino acid sequences of approximately 500–600 residues, and subtypes share between 30% and 65% identity; typically greater than 80% identity is shared when comparing a particular subtype across species. Together with two structurally related neutral amino acid transporter genes (alanine/serine/ cysteine transporter genes ASCT1–ASCT2), they make up a gene superfamily whose members are structurally distinct from other solute transporters, such as the vesicular glutamate transporters (GLTs) and the family of Naþ/Cl-dependent neurotransmitter transporters responsible for uptake of GABA, glycine, serotonin, and catecholamines. Distinct EAAT subtypes are found on neurons and glia throughout the brain and spinal cord, and different functional roles are emerging for each. In the central nervous system (CNS), the vast majority of glutamate uptake activity is found on glial cell membranes (predominantly EAAT2/GLT-1 in forebrain and spinal cord, and EAAT1/GLAST in cerebellum). These glial transporters are densely packed in the membrane (up to 8500 mm2) and are often found in close apposition to neuronal synapses. Table 1 provides an overview of the nomenclature and expression patterns of the transporters.
(HP1 and HP2) that dip into the membrane from opposite sides. Structural as well as functional evidence suggests that each subunit in the trimer binds substrate: in the crystal structure a molecule of bound aspartate is occluded between the tips of the two reentrant loops, with Naþ ions above and below it. In an apo-state transporter crystallized without bound amino acid, the diffraction pattern reveals that the external reentrant loop (HP2) is swung outward approximately 10 A˚, suggesting a possible docking trajectory and structural gating mechanism for the first hemicycle of an alternating access kinetic scheme (see later). In the occluded state, charge pairing between the bound aspartate and residues in the transporter stabilize the complex. A key determinant of substrate selectivity is a conserved arginine residue in transmembrane domain 8 (TMD8) that is coordinated with the distal carboxylate of the transported substrate (Figure 1). In the related ASC transporters, which selectively transport neutral rather than acidic amino acids, a cysteine or threonine residue is found in the analogous position in TMD8. Mutating the neutral residue to arginine in the ASC transporters or making the converse mutation in the EAATs switches the transporters’ selectivity correspondingly. All of the EAATs are stereoselective for L-glutamate, while both enantiomers of aspartate are transported. A number of glutamate and aspartate analogs, including some ring-constrained analogs such as pyrrolidine dicarboxylates (PDCs), are transported by the EAATs. More bulky aromatic analogs such as DL-threob-benzyloxyaspartate (TBOA) competitively inhibit glutamate transport but are not themselves transported. With the exception of kainate and dihydrokainate, which block EAAT2 with an affinity three to four orders of magnitude higher than the other transporter isoforms, there are no compounds available with which to distinguish the transporter subtypes with a high degree of selectivity in vivo.
Thermodynamics of Glutamate Transport Molecular Structure GltPh is an aspartate/glutamate transporter homolog from Pyroccocus horikoshii that exhibits approximately 35% identity to mammalian EAATs overall, with higher homology in domains that have been implicated in key functions such as substrate and ion binding. The three-dimensional structure of GltPh has been determined by X-ray crystallography, revealing a rather complex trimeric bowl-shaped architecture. Each of the three subunits has eight transmembrane domains with two reentrant helical hairpin loops
The influx of Naþ and Hþ and the efflux of Kþ during glutamate uptake result in a net flow of positive charge into the cell that can be recorded and studied with a voltage-clamp circuit. A tight stoichiometric coupling is inferred from the effects of these cations’ concentration gradients on the reversal potential of the pharmacologically isolated transport current. The transporter reversal potential (the equilibrium membrane potential at which there is no net transport) follows the predictions of the free energy equation for coupled transport of one glutamate molecule
806 Glial Glutamate Transporters: Electrophysiology Table 1 Glutamate transporter nomenclature and expression patterns Human
Other
IUBMB nomenclaturea
Predominant expression pattern
EAAT1 EAAT2
GLAST GLT-1
SLC1A3 SLC1A2
EAAT3
EAAC1
SLC1A1
EAAT4 EAAT5
EAAT4 EAAT5
SLC1A4 SLC1A5
Astrocytic; cerebellum (Bergmann glia); retina (Mu¨ller cells); inner ear Astrocytic (most abundant transporter); brain and spinal cord, low levels in some neuron terminals Predominantly neuronal (widely expressed but levels much lower than EAAT1/2); predominant peripheral glutamate transporter Neuronal; cerebellum (Purkinje neurons) Retina: photoreceptors and bipolar cells, astrocytes (Mu¨ller cells)
a
International Union of Biochemistry and Molecular Biology.
Arg
Asp
HP2
Figure 1 Superimposed structures of the Pyroccocus horikoshii aspartate/glutamate transporter homolog transport states (see text for discussion) To (green) and Tocc (blue), highlighting translation of hairpin 2 (HP2) loop going from empty To state to the occluded state. Bound substrate (aspartate) interaction with arginine residue in transmembrane domain 8 is shown, with two Naþ ions (spheres) below. Python-enhanced molecular graphics tool representation of Protein Data Bank coordinates 2NWX and 2NWW.
with one proton and three Naþ ions and countertransport of one Kþ ion during each uptake cycle. The equilibrium glutamate concentration gradient that can be maintained by stoichiometrically coupling movement of n Naþ, Kþ, and Hþ ions is given by rearrangement of the zero-flux equation: ðGluo =Glui Þ ¼ ðNai =Nao ÞnNa ðHi =Ho ÞnH ðKo =Ki ÞnK exp½ðnNa þ nH nGlu nK ÞVF=RT
½1
where V¼ membrane potential, F ¼ Faraday’s constant, R ¼ gas constant, and T ¼ temperature. For astrocytes and neurons, this value exceeds 106; the free energy in the Naþ, Kþ, and Hþ electrochemical gradients is sufficient to maintain nanomolar extracellular glutamate concentrations despite millimolar intracellular glutamate concentrations. Estimates of extracellular glutamate concentrations using dialysis
probes in brain tissue suggest low micromolar values, but electrophysiological measurements in hippocampus are consistent with ambient glutamate levels below 100 nM. When ionic gradients are impaired, with elevated extracellular Kþ and intracellular Naþ, as can occur during severe ischemia, transporter reversal and efflux of intracellular glutamate may occur. Another distinct mode of transport, electroneutral glutamate exchange, can occur in the absence of Kþ when Naþ and glutamate (or another substrate) are present on both sides of the membrane. This exchange can be monitored by measuring the unidirectional flux of radioactivity when radiolabeled substrate is added to one side of the membrane. The glutamate exchange mode can also be induced by mutations in the transporter near the substrate binding site that selectively interfere with Kþ binding (e.g., E404D; GLT-1 numbering).
Mechanism and Kinetics Mechanistically, the observed transport modes are consistent with high-resolution glutamate transporter structures that appear to represent distinct kinetic states. Together, the structural and functional data suggest that the transporter cycles through three major states: one occluded state (Tocc), and two distinct states (To and Ti) that provide gated access of substrates to their binding sites from outside or inside the cell, respectively. The structures of the first two of these states have been solved (Figure 1) and the existence of the third state (Ti) is only inferred from kinetic data at this point. There is no evidence for a state with outer and inner gates simultaneously open (i.e., an open channel permeable to glutamate). Access for intra- and extracellular substrates appears to occur on a strictly alternating basis, providing a basis for tight thermodynamic coupling of glutamate and cation fluxes. A further tenet of the model supported by functional data is that Naþ and Kþ binding are mutually exclusive. Naþ and glutamate binding will occur in a coordinated fashion and their
Glial Glutamate Transporters: Electrophysiology
occupancy precludes binding of a Kþ ion. Conversely, binding of a Kþ ion precludes Naþ and glutamate binding. Kinetic evidence, primarily from the Naþ and glutamate concentration-dependence of mammalian EAATs, suggests that binding of two Naþ ions precedes glutamate binding, followed by the third Naþ ion. The postulated sequence of events for one thermodynamic cycle (arbitrarily starting with the glutamate hemicycle) is as follows. Glutamate/Naþ/ Hþ bind from outside (or inside) to a transporter in state To or Ti, respectively, thereby preventing binding of a competing Kþ ion. The two major states, To and Ti, are kinetically linked to each other through the occluded state. Unbinding of glutamate/Naþ/Hþ can occur from either state To or Ti after transition out of the occluded state; then a competition with Kþ for rebinding occurs. For a complete transport cycle to occur, the first hemicycle is completed when glutamate/Naþ/Hþ are translocated by unbinding from the side opposite to which they bound. A Kþ ion then binds and proceeds back through the same kinetic steps, completing the second hemicycle by its unbinding from the opposite side and restoring the initial state. The free energy change for the cycle is equal to the sum of the free energy changes for the individual ions. All of these kinetic transitions are reversible; the net flow through the states depends only on the transition rate constants, the concentration gradients for Naþ, glutamate, Hþ, and Kþ, and the membrane potential. The conformational transition of the unliganded transporter between the Ti and To states does not appear to proceed at a measurable rate; possibly occupancy by substrate is necessary to stabilize an unfavorable transition state. Overall, the mechanism bears notable similarity to the Na/K pump, with the primary difference being that the vectorial nature of transport in this case is a consequence of electrochemical gradients rather than ATP hydrolysis. Studies of glutamate transport kinetics have utilized both steady-state and pre-steady-state techniques. The latter include analysis of transient currents following rapid concentration or voltage jumps. In transporters covalently labeled with fluorescent reporter probes, fluorescence changes can also be measured in response to perturbations of voltage or concentration. A prominent capacitive current (or fluorescence change) reflecting charge movements is present in the transporters that is analogous to gating charge movements seen in voltage-gated channels. This charge movement is Naþ dependent, and it occurs in the absence of glutamate, providing evidence that Naþ binding occurs prior to glutamate. The transient current or fluorescence change can be described by a Boltzmann function, similar to channel gating charge
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movements, and in the case of the transient current it can be used to estimate the number of transporters in a cell or patch. Using this approach, the turnover rate of the major glial transporter EAAT2 expressed in Xenopus oocytes is approximately 15 s1 at room temperature, or approximately 50 s1 at 37 , because of the transporter’s temperature dependence. Because many rates in the transport cycle are voltage dependent, the turnover rate is also accelerated by hyperpolarizing membrane potentials (approximately e-fold per 75 mV in the case of EAAT2). Other techniques used to gain information about microscopic transport kinetics include rapid photo-uncaging or piezoswitched application of glutamate to cells or excised patches containing transporters. In the presence of extracellular Naþ and intracellular Kþ, a glutamate pulse produces a fast inward current that rapidly relaxes to a lower steady-state level (Figure 2(a)). This reflects fast glutamate binding (107M1s1) and rapid charge movements across the electric field in the early steps of the transport cycle. Using paired concentration jumps applied to excised patches to measure the kinetics of recovery of the transient peak current, EAAT2 turnover rates exceeding 50 s1 have been estimated. This faster turnover (compared with steady-state oocyte measurements) likely reflects the absence of intracellular Naþ and glutamate, which will slow the transport cycle in oocytes (as well as glia and neurons) because of the aforementioned microscopic kinetics. Even at 20 ms, the transport cycle time is roughly an order of magnitude slower than the estimated synaptic glutamate concentration decay. Nevertheless, the transporters near a release site may still theoretically influence the spatiotemporal profile of glutamate on a very fast timescale because the early steps in the transport cycle allow rapid glutamate capture and the transporters are present at high density
10 pA a
b
50 ms
Figure 2 Outside-out patch recordings of glial transporter EAAT2, showing anion channel and glutamate transport kinetic responses to piezo-switched application of saturating (10 mM) glutamate. (a) Response predominantly reflects stoichiometrically coupled currents; (b) reflects anion currents carried by SCN. Pipette solution contained K-gluconate; bath contained NaCl (a) or NaSCN (b). Data are from the same patch held at 20 mV; trace above shows solution exchange time course measured following patch rupture.
808 Glial Glutamate Transporters: Electrophysiology
(103–104 mm2) on astrocyte membranes. Furthermore, more remote transporters surrounding synaptic release sites can play roles in restricting the heterosynaptic spillover of glutamate that occurs on a slower timescale (see later).
Chloride Channel Function The aforementioned fluxes of stoichiometrically coupled ions through the transporter are not the only charge movements mediated by EAATs; chloride flux through the transporters also occurs. This was first suggested by the presence of a chloride current associated with activation of the native glutamate transporters in retinal neurons, and it has subsequently been demonstrated to occur with EAATs exogenously expressed in many different cell systems. Interestingly, this channel function can play roles in synaptic signaling that are entirely distinct from the effects of uptake on glutamate dynamics. Glutamate transporters (predominantly EAAT5) on the presynaptic terminals of rod bipolar cells mediate inhibitory signaling that serves to modulate the release of transmitter in a negative feedback loop. Several lines of evidence suggest that a chloride channel is intrinsic to all glutamate transporters. Mutation of residues clustered in TMD2 leads to discrete changes in anion selectivity. In addition, the net current activated by glutamate (reflecting both the stoichiometrically coupled and Cl currents) has a distinct reversal potential in each EAAT isoform, suggesting that each has a fixed and unique Cl current magnitude relative to the stoichiometrically coupled current. The relative magnitude of anion conductance to coupled current follows the sequence: EAAT4 EAAT5 > EAAT1 > EAAT3 > EAAT2. In each transporter, the anion conductance displays a chaotropic selectivity sequence (e.g., SCN is approximately 70-fold more permeant than Cl is in EAAT1). The magnitude of the anion conductance through a particular EAAT is also substrate specific; for example, D-aspartate activates a relatively larger anion conductance through EAAT1 than does L-glutamate, resulting in different net current reversal potentials for these substrates. In the absence of glutamate, a small tonic anion leak is present that seems to require Naþ. The glutamatedependent anion conductance is also affected by the identity of the alkali cation cotransported with glutamate; Liþ can substitute for Naþ in some isoforms to support glutamate transport but is much less efficacious at activating the anion conductance. Importantly, in at least some of the EAAT isoforms, replacement of Cl with impermeant anions like gluconate does not affect glutamate flux. Cl flux thus is thermodynamically uncoupled from glutamate flux,
and may involve a channel-like mechanism instead. Indeed, glutamate-dependent anion current fluctuations have been observed in whole-cell and excised patch recordings that are consistent with a stochastically gated channel mechanism related to but distinct from the alternating-access glutamate transport mechanism. Comparative analysis of the glutamate concentration dependence of both transport and chloride channel gating suggests that each subunit in the trimer harbors both a chloride channel and a glutamate transporter; furthermore, each subunit functions independent of the other two subunits. Activation of the anion current in response to rapid glutamate application reveals similar but somewhat slower dynamics than does the stoichiometrically coupled current (Figure 2). The structural determinants and kinetics of the anion channel and the transporter may indeed be intimately related despite the important differences between channel and carrier gating. Kinetic models may be able to reconcile these differences by representing a subset of the Markov states in the transport cycle as open-channel states. Covalent modification of several mutants with engineered cysteine residues (e.g., V417C; EAAC1 numbering) eliminates transport but does not prevent activation of the anion current by extracellular glutamate, suggesting that some or all of the open-channel states may be in the To or Tocc forms.
Synaptic Physiology As previously discussed, the coupled fluxes of glutamate, Naþ, Hþ, and Kþ would theoretically allow maintenance of ambient extracellular glutamate levels in the low nanomolar concentration range. However, estimates of actual glutamate levels in the brain tend to be significantly higher, and in some areas are above the threshold required to generate tonic activity of N-methyl-D-aspartate (NMDA) and/or metabotropic glutamate receptors. The tonic extracellular level reflects a nonequilibrium state determined by the rates of uptake and release. The latter includes both synaptic and nonsynaptic components. Factors influencing the extracellular glutamate concentration include the density of EAATs and the frequency of synaptic activity. Nonsynaptic routes of release, particularly from astrocytes, also contribute to the extracellular glutamate level. The nonsynaptic mechanisms are generally less well characterized, but appear to include swelling-activated anion channels, the system xc- glutamate–cystine exchanger, and possibly other routes. In addition to playing a central role in determining ambient glutamate levels, EAAT activity can also influence the temporal concentration profile of synaptically
Glial Glutamate Transporters: Electrophysiology
released glutamate. Following vesicle fusion, the glutamate concentration in the synapse briefly reaches millimolar values. The dynamics of the concentration decay are strongly influenced by the geometry of the synapse and surrounding barriers. Diffusion appears to account for the major component of clearance within the synapse, with a time constant of a few milliseconds. However, EAAT activity can also influence the kinetics of the postsynaptic response in a manner dependent not only on the synaptic and extrasynaptic geometry, but also on receptor affinity, receptor distance from release sites, and release frequency. Additionally, multivesicular release and/or heterosynaptic glutamate spillover between release sites can result in slower transmitter clearance and a larger relative contribution of EAATs to transmitter decay kinetics. There is a great deal of heterogeneity in the effect of transporter blockade on synaptic transmission at different synapses. In general, the influence of uptake on synaptic responses becomes more pronounced as postsynaptic receptor affinity for glutamate increases, receptor kinetics slow, and proximity to the release site decreases. Oftentimes there is a spectrum of effects on different components of the synaptic response according to these criteria. Thus, the response kinetics of a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors within the synapse tend to be less influenced by glutamate transport than do the responses of extrasynaptic NMDA or metabotropic glutamate receptors. Synapse organization in the CNS exhibits a rich diversity, and the contributions of EAATs to synaptic signaling are correspondingly varied.
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See also: Glial Glutamate Transporters; Glial Glutamate and GABA Metabolism; Glutamate; Glutamate Receptor Organization: Ultrastructural Insights; Ionic Channels in Glia.
Further Reading Boudker O, Ryan RM, Yernool D, et al. (2007) Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445: 387–393. Brasno G and Otis TS (2001) Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression. Neuron 31: 607–616. Christie JM and Jahr CE (2006) Multivesicular release at Schaffer collateral–CA1 hippocampal synapses. Journal of Neuroscience 26: 210–216. Danbolt N (2002) Glutamate uptake. Progress in Neurobiology 65: 1–105. Grewer C and Rauen T (2005) Electrogenic glutamate transporters in the CNS: Molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling. Journal of Membrane Biology 203: 1–20. Huang YH and Bergles DE (2004) Glutamate transporters bring competition to the synapse. Current Opinion in Neurobiology 14: 346–352. Zerangue N and Kavanaugh MP (1996) Flux coupling in a neuronal glutamate transporter. Nature 383: 634–637.
Relevant Websites http://www.rcsb.org – Protein Data Bank (Research Collaboratory for Structural Bioinformatics). http://sourceforge.net – Sourceforge.net. Python-enhanced molecular graphics tool.
805
Glial Glutamate Transporters: Electrophysiology M P Kavanaugh, University of Montana, Missoula, MT, USA ã 2009 Elsevier Ltd. All rights reserved.
Molecular Diversity and Expression Patterns The mammalian glutamate transporter gene family has five excitatory amino acid transporter (EAAT) members (EAAT1–EAAT5) that are expressed in the brain in highly specific regional and cell-type patterns (see Table 1). In some cases, transporter transcripts undergo alternative splicing, adding further complexity to these expression patterns. EAAT cDNAs have predicted amino acid sequences of approximately 500–600 residues, and subtypes share between 30% and 65% identity; typically greater than 80% identity is shared when comparing a particular subtype across species. Together with two structurally related neutral amino acid transporter genes (alanine/serine/ cysteine transporter genes ASCT1–ASCT2), they make up a gene superfamily whose members are structurally distinct from other solute transporters, such as the vesicular glutamate transporters (GLTs) and the family of Naþ/Cl-dependent neurotransmitter transporters responsible for uptake of GABA, glycine, serotonin, and catecholamines. Distinct EAAT subtypes are found on neurons and glia throughout the brain and spinal cord, and different functional roles are emerging for each. In the central nervous system (CNS), the vast majority of glutamate uptake activity is found on glial cell membranes (predominantly EAAT2/GLT-1 in forebrain and spinal cord, and EAAT1/GLAST in cerebellum). These glial transporters are densely packed in the membrane (up to 8500 mm2) and are often found in close apposition to neuronal synapses. Table 1 provides an overview of the nomenclature and expression patterns of the transporters.
(HP1 and HP2) that dip into the membrane from opposite sides. Structural as well as functional evidence suggests that each subunit in the trimer binds substrate: in the crystal structure a molecule of bound aspartate is occluded between the tips of the two reentrant loops, with Naþ ions above and below it. In an apo-state transporter crystallized without bound amino acid, the diffraction pattern reveals that the external reentrant loop (HP2) is swung outward approximately 10 A˚, suggesting a possible docking trajectory and structural gating mechanism for the first hemicycle of an alternating access kinetic scheme (see later). In the occluded state, charge pairing between the bound aspartate and residues in the transporter stabilize the complex. A key determinant of substrate selectivity is a conserved arginine residue in transmembrane domain 8 (TMD8) that is coordinated with the distal carboxylate of the transported substrate (Figure 1). In the related ASC transporters, which selectively transport neutral rather than acidic amino acids, a cysteine or threonine residue is found in the analogous position in TMD8. Mutating the neutral residue to arginine in the ASC transporters or making the converse mutation in the EAATs switches the transporters’ selectivity correspondingly. All of the EAATs are stereoselective for L-glutamate, while both enantiomers of aspartate are transported. A number of glutamate and aspartate analogs, including some ring-constrained analogs such as pyrrolidine dicarboxylates (PDCs), are transported by the EAATs. More bulky aromatic analogs such as DL-threob-benzyloxyaspartate (TBOA) competitively inhibit glutamate transport but are not themselves transported. With the exception of kainate and dihydrokainate, which block EAAT2 with an affinity three to four orders of magnitude higher than the other transporter isoforms, there are no compounds available with which to distinguish the transporter subtypes with a high degree of selectivity in vivo.
Thermodynamics of Glutamate Transport Molecular Structure GltPh is an aspartate/glutamate transporter homolog from Pyroccocus horikoshii that exhibits approximately 35% identity to mammalian EAATs overall, with higher homology in domains that have been implicated in key functions such as substrate and ion binding. The three-dimensional structure of GltPh has been determined by X-ray crystallography, revealing a rather complex trimeric bowl-shaped architecture. Each of the three subunits has eight transmembrane domains with two reentrant helical hairpin loops
The influx of Naþ and Hþ and the efflux of Kþ during glutamate uptake result in a net flow of positive charge into the cell that can be recorded and studied with a voltage-clamp circuit. A tight stoichiometric coupling is inferred from the effects of these cations’ concentration gradients on the reversal potential of the pharmacologically isolated transport current. The transporter reversal potential (the equilibrium membrane potential at which there is no net transport) follows the predictions of the free energy equation for coupled transport of one glutamate molecule
806 Glial Glutamate Transporters: Electrophysiology Table 1 Glutamate transporter nomenclature and expression patterns Human
Other
IUBMB nomenclaturea
Predominant expression pattern
EAAT1 EAAT2
GLAST GLT-1
SLC1A3 SLC1A2
EAAT3
EAAC1
SLC1A1
EAAT4 EAAT5
EAAT4 EAAT5
SLC1A4 SLC1A5
Astrocytic; cerebellum (Bergmann glia); retina (Mu¨ller cells); inner ear Astrocytic (most abundant transporter); brain and spinal cord, low levels in some neuron terminals Predominantly neuronal (widely expressed but levels much lower than EAAT1/2); predominant peripheral glutamate transporter Neuronal; cerebellum (Purkinje neurons) Retina: photoreceptors and bipolar cells, astrocytes (Mu¨ller cells)
a
International Union of Biochemistry and Molecular Biology.
Arg
Asp
HP2
Figure 1 Superimposed structures of the Pyroccocus horikoshii aspartate/glutamate transporter homolog transport states (see text for discussion) To (green) and Tocc (blue), highlighting translation of hairpin 2 (HP2) loop going from empty To state to the occluded state. Bound substrate (aspartate) interaction with arginine residue in transmembrane domain 8 is shown, with two Naþ ions (spheres) below. Python-enhanced molecular graphics tool representation of Protein Data Bank coordinates 2NWX and 2NWW.
with one proton and three Naþ ions and countertransport of one Kþ ion during each uptake cycle. The equilibrium glutamate concentration gradient that can be maintained by stoichiometrically coupling movement of n Naþ, Kþ, and Hþ ions is given by rearrangement of the zero-flux equation: ðGluo =Glui Þ ¼ ðNai =Nao ÞnNa ðHi =Ho ÞnH ðKo =Ki ÞnK exp½ðnNa þ nH nGlu nK ÞVF=RT
½1
where V¼ membrane potential, F ¼ Faraday’s constant, R ¼ gas constant, and T ¼ temperature. For astrocytes and neurons, this value exceeds 106; the free energy in the Naþ, Kþ, and Hþ electrochemical gradients is sufficient to maintain nanomolar extracellular glutamate concentrations despite millimolar intracellular glutamate concentrations. Estimates of extracellular glutamate concentrations using dialysis
probes in brain tissue suggest low micromolar values, but electrophysiological measurements in hippocampus are consistent with ambient glutamate levels below 100 nM. When ionic gradients are impaired, with elevated extracellular Kþ and intracellular Naþ, as can occur during severe ischemia, transporter reversal and efflux of intracellular glutamate may occur. Another distinct mode of transport, electroneutral glutamate exchange, can occur in the absence of Kþ when Naþ and glutamate (or another substrate) are present on both sides of the membrane. This exchange can be monitored by measuring the unidirectional flux of radioactivity when radiolabeled substrate is added to one side of the membrane. The glutamate exchange mode can also be induced by mutations in the transporter near the substrate binding site that selectively interfere with Kþ binding (e.g., E404D; GLT-1 numbering).
Mechanism and Kinetics Mechanistically, the observed transport modes are consistent with high-resolution glutamate transporter structures that appear to represent distinct kinetic states. Together, the structural and functional data suggest that the transporter cycles through three major states: one occluded state (Tocc), and two distinct states (To and Ti) that provide gated access of substrates to their binding sites from outside or inside the cell, respectively. The structures of the first two of these states have been solved (Figure 1) and the existence of the third state (Ti) is only inferred from kinetic data at this point. There is no evidence for a state with outer and inner gates simultaneously open (i.e., an open channel permeable to glutamate). Access for intra- and extracellular substrates appears to occur on a strictly alternating basis, providing a basis for tight thermodynamic coupling of glutamate and cation fluxes. A further tenet of the model supported by functional data is that Naþ and Kþ binding are mutually exclusive. Naþ and glutamate binding will occur in a coordinated fashion and their
Glial Glutamate Transporters: Electrophysiology
occupancy precludes binding of a Kþ ion. Conversely, binding of a Kþ ion precludes Naþ and glutamate binding. Kinetic evidence, primarily from the Naþ and glutamate concentration-dependence of mammalian EAATs, suggests that binding of two Naþ ions precedes glutamate binding, followed by the third Naþ ion. The postulated sequence of events for one thermodynamic cycle (arbitrarily starting with the glutamate hemicycle) is as follows. Glutamate/Naþ/ Hþ bind from outside (or inside) to a transporter in state To or Ti, respectively, thereby preventing binding of a competing Kþ ion. The two major states, To and Ti, are kinetically linked to each other through the occluded state. Unbinding of glutamate/Naþ/Hþ can occur from either state To or Ti after transition out of the occluded state; then a competition with Kþ for rebinding occurs. For a complete transport cycle to occur, the first hemicycle is completed when glutamate/Naþ/Hþ are translocated by unbinding from the side opposite to which they bound. A Kþ ion then binds and proceeds back through the same kinetic steps, completing the second hemicycle by its unbinding from the opposite side and restoring the initial state. The free energy change for the cycle is equal to the sum of the free energy changes for the individual ions. All of these kinetic transitions are reversible; the net flow through the states depends only on the transition rate constants, the concentration gradients for Naþ, glutamate, Hþ, and Kþ, and the membrane potential. The conformational transition of the unliganded transporter between the Ti and To states does not appear to proceed at a measurable rate; possibly occupancy by substrate is necessary to stabilize an unfavorable transition state. Overall, the mechanism bears notable similarity to the Na/K pump, with the primary difference being that the vectorial nature of transport in this case is a consequence of electrochemical gradients rather than ATP hydrolysis. Studies of glutamate transport kinetics have utilized both steady-state and pre-steady-state techniques. The latter include analysis of transient currents following rapid concentration or voltage jumps. In transporters covalently labeled with fluorescent reporter probes, fluorescence changes can also be measured in response to perturbations of voltage or concentration. A prominent capacitive current (or fluorescence change) reflecting charge movements is present in the transporters that is analogous to gating charge movements seen in voltage-gated channels. This charge movement is Naþ dependent, and it occurs in the absence of glutamate, providing evidence that Naþ binding occurs prior to glutamate. The transient current or fluorescence change can be described by a Boltzmann function, similar to channel gating charge
807
movements, and in the case of the transient current it can be used to estimate the number of transporters in a cell or patch. Using this approach, the turnover rate of the major glial transporter EAAT2 expressed in Xenopus oocytes is approximately 15 s1 at room temperature, or approximately 50 s1 at 37 , because of the transporter’s temperature dependence. Because many rates in the transport cycle are voltage dependent, the turnover rate is also accelerated by hyperpolarizing membrane potentials (approximately e-fold per 75 mV in the case of EAAT2). Other techniques used to gain information about microscopic transport kinetics include rapid photo-uncaging or piezoswitched application of glutamate to cells or excised patches containing transporters. In the presence of extracellular Naþ and intracellular Kþ, a glutamate pulse produces a fast inward current that rapidly relaxes to a lower steady-state level (Figure 2(a)). This reflects fast glutamate binding (107M1s1) and rapid charge movements across the electric field in the early steps of the transport cycle. Using paired concentration jumps applied to excised patches to measure the kinetics of recovery of the transient peak current, EAAT2 turnover rates exceeding 50 s1 have been estimated. This faster turnover (compared with steady-state oocyte measurements) likely reflects the absence of intracellular Naþ and glutamate, which will slow the transport cycle in oocytes (as well as glia and neurons) because of the aforementioned microscopic kinetics. Even at 20 ms, the transport cycle time is roughly an order of magnitude slower than the estimated synaptic glutamate concentration decay. Nevertheless, the transporters near a release site may still theoretically influence the spatiotemporal profile of glutamate on a very fast timescale because the early steps in the transport cycle allow rapid glutamate capture and the transporters are present at high density
10 pA a
b
50 ms
Figure 2 Outside-out patch recordings of glial transporter EAAT2, showing anion channel and glutamate transport kinetic responses to piezo-switched application of saturating (10 mM) glutamate. (a) Response predominantly reflects stoichiometrically coupled currents; (b) reflects anion currents carried by SCN. Pipette solution contained K-gluconate; bath contained NaCl (a) or NaSCN (b). Data are from the same patch held at 20 mV; trace above shows solution exchange time course measured following patch rupture.
808 Glial Glutamate Transporters: Electrophysiology
(103–104 mm2) on astrocyte membranes. Furthermore, more remote transporters surrounding synaptic release sites can play roles in restricting the heterosynaptic spillover of glutamate that occurs on a slower timescale (see later).
Chloride Channel Function The aforementioned fluxes of stoichiometrically coupled ions through the transporter are not the only charge movements mediated by EAATs; chloride flux through the transporters also occurs. This was first suggested by the presence of a chloride current associated with activation of the native glutamate transporters in retinal neurons, and it has subsequently been demonstrated to occur with EAATs exogenously expressed in many different cell systems. Interestingly, this channel function can play roles in synaptic signaling that are entirely distinct from the effects of uptake on glutamate dynamics. Glutamate transporters (predominantly EAAT5) on the presynaptic terminals of rod bipolar cells mediate inhibitory signaling that serves to modulate the release of transmitter in a negative feedback loop. Several lines of evidence suggest that a chloride channel is intrinsic to all glutamate transporters. Mutation of residues clustered in TMD2 leads to discrete changes in anion selectivity. In addition, the net current activated by glutamate (reflecting both the stoichiometrically coupled and Cl currents) has a distinct reversal potential in each EAAT isoform, suggesting that each has a fixed and unique Cl current magnitude relative to the stoichiometrically coupled current. The relative magnitude of anion conductance to coupled current follows the sequence: EAAT4 EAAT5 > EAAT1 > EAAT3 > EAAT2. In each transporter, the anion conductance displays a chaotropic selectivity sequence (e.g., SCN is approximately 70-fold more permeant than Cl is in EAAT1). The magnitude of the anion conductance through a particular EAAT is also substrate specific; for example, D-aspartate activates a relatively larger anion conductance through EAAT1 than does L-glutamate, resulting in different net current reversal potentials for these substrates. In the absence of glutamate, a small tonic anion leak is present that seems to require Naþ. The glutamatedependent anion conductance is also affected by the identity of the alkali cation cotransported with glutamate; Liþ can substitute for Naþ in some isoforms to support glutamate transport but is much less efficacious at activating the anion conductance. Importantly, in at least some of the EAAT isoforms, replacement of Cl with impermeant anions like gluconate does not affect glutamate flux. Cl flux thus is thermodynamically uncoupled from glutamate flux,
and may involve a channel-like mechanism instead. Indeed, glutamate-dependent anion current fluctuations have been observed in whole-cell and excised patch recordings that are consistent with a stochastically gated channel mechanism related to but distinct from the alternating-access glutamate transport mechanism. Comparative analysis of the glutamate concentration dependence of both transport and chloride channel gating suggests that each subunit in the trimer harbors both a chloride channel and a glutamate transporter; furthermore, each subunit functions independent of the other two subunits. Activation of the anion current in response to rapid glutamate application reveals similar but somewhat slower dynamics than does the stoichiometrically coupled current (Figure 2). The structural determinants and kinetics of the anion channel and the transporter may indeed be intimately related despite the important differences between channel and carrier gating. Kinetic models may be able to reconcile these differences by representing a subset of the Markov states in the transport cycle as open-channel states. Covalent modification of several mutants with engineered cysteine residues (e.g., V417C; EAAC1 numbering) eliminates transport but does not prevent activation of the anion current by extracellular glutamate, suggesting that some or all of the open-channel states may be in the To or Tocc forms.
Synaptic Physiology As previously discussed, the coupled fluxes of glutamate, Naþ, Hþ, and Kþ would theoretically allow maintenance of ambient extracellular glutamate levels in the low nanomolar concentration range. However, estimates of actual glutamate levels in the brain tend to be significantly higher, and in some areas are above the threshold required to generate tonic activity of N-methyl-D-aspartate (NMDA) and/or metabotropic glutamate receptors. The tonic extracellular level reflects a nonequilibrium state determined by the rates of uptake and release. The latter includes both synaptic and nonsynaptic components. Factors influencing the extracellular glutamate concentration include the density of EAATs and the frequency of synaptic activity. Nonsynaptic routes of release, particularly from astrocytes, also contribute to the extracellular glutamate level. The nonsynaptic mechanisms are generally less well characterized, but appear to include swelling-activated anion channels, the system xc- glutamate–cystine exchanger, and possibly other routes. In addition to playing a central role in determining ambient glutamate levels, EAAT activity can also influence the temporal concentration profile of synaptically
Glial Glutamate Transporters: Electrophysiology
released glutamate. Following vesicle fusion, the glutamate concentration in the synapse briefly reaches millimolar values. The dynamics of the concentration decay are strongly influenced by the geometry of the synapse and surrounding barriers. Diffusion appears to account for the major component of clearance within the synapse, with a time constant of a few milliseconds. However, EAAT activity can also influence the kinetics of the postsynaptic response in a manner dependent not only on the synaptic and extrasynaptic geometry, but also on receptor affinity, receptor distance from release sites, and release frequency. Additionally, multivesicular release and/or heterosynaptic glutamate spillover between release sites can result in slower transmitter clearance and a larger relative contribution of EAATs to transmitter decay kinetics. There is a great deal of heterogeneity in the effect of transporter blockade on synaptic transmission at different synapses. In general, the influence of uptake on synaptic responses becomes more pronounced as postsynaptic receptor affinity for glutamate increases, receptor kinetics slow, and proximity to the release site decreases. Oftentimes there is a spectrum of effects on different components of the synaptic response according to these criteria. Thus, the response kinetics of a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors within the synapse tend to be less influenced by glutamate transport than do the responses of extrasynaptic NMDA or metabotropic glutamate receptors. Synapse organization in the CNS exhibits a rich diversity, and the contributions of EAATs to synaptic signaling are correspondingly varied.
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See also: Glial Glutamate Transporters; Glial Glutamate and GABA Metabolism; Glutamate; Glutamate Receptor Organization: Ultrastructural Insights; Ionic Channels in Glia.
Further Reading Boudker O, Ryan RM, Yernool D, et al. (2007) Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445: 387–393. Brasno G and Otis TS (2001) Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression. Neuron 31: 607–616. Christie JM and Jahr CE (2006) Multivesicular release at Schaffer collateral–CA1 hippocampal synapses. Journal of Neuroscience 26: 210–216. Danbolt N (2002) Glutamate uptake. Progress in Neurobiology 65: 1–105. Grewer C and Rauen T (2005) Electrogenic glutamate transporters in the CNS: Molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling. Journal of Membrane Biology 203: 1–20. Huang YH and Bergles DE (2004) Glutamate transporters bring competition to the synapse. Current Opinion in Neurobiology 14: 346–352. Zerangue N and Kavanaugh MP (1996) Flux coupling in a neuronal glutamate transporter. Nature 383: 634–637.
Relevant Websites http://www.rcsb.org – Protein Data Bank (Research Collaboratory for Structural Bioinformatics). http://sourceforge.net – Sourceforge.net. Python-enhanced molecular graphics tool.