glutamate rev
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The presence of an extensive array of excitatoryamino acid (EAA) ionotropic and metabotropicreceptors is critical to the ability of theneurotransmitter -glutamate to contribute to aspectrum of functions in the CNS that ranges fromstandard fast synaptic signaling to neuroplasticity toneurodegeneration. Another importantelement in this equation, however, is the amount of
-glutamate present in the synaptic environment andit is within this context that there is increasing interestin the glutamate transport systems. The capacity of these uptake systems to effectively sequester
-glutamate in glia, neurons and in synaptic vesiclesis likely to be an important factor in the release, signaltermination, and recycling of this excitatory
neurotransmitter, as well as in the protection of neurons from excitotoxic injury. Thegoal of this brief review is to provide an update on anumber of the pharmacological tools that are being
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Introduction
used to define and manipulate the function of theseglutamate transporters.
While -glutamate can act as a substrate of numerouscellular uptake systems, the transporters accepted asplaying the most significant role in relation toexcitatory neurotransmission in the CNS arecollectively referred to as the high-affinity, sodium-dependent systems. Harnessing transmembrane iongradients as the driving force, these uptake systemsare capable of maintaining intracellular concentrationsof -glutamate several thousand-fold aboveextracellular levels. Stoichiometric studies suggestthat the intracellular translocation of one molecule of
-glutamate is coupled to the inward movement of 3Na ions and 1 H ion and the outward movement of 1K ion. Comparisons of substrate specificity (e.g.glutamate vs. homocysteate), differential inhibition(e.g. sensitivity to dihydrokainate), kinetics, cell type(e.g. neuron vs. glia) or anatomical location (e.g.cortical vs. cerebellar) in a variety of preparations allpointed to heterogeneity within the sodium-dependenttransport proteins. A major step, not only resolvingthis issue, but in advancing our understanding of thestructure, mechanism and physiological properties of the transporters, came with the cloning andexpression of distinct transporter subtypes.Referred to as EAATs (used for the human clones asan acronym for xcitatory mino cid ransporters),at least 5 distinct subtypes have been identified that
(along with the neutral amino acid transporters ASCT1 and ASCT2) appear to be part of a novelgene family. A conserved heptapeptide sequence[AA(I/Q)FIAQ] appears to be a defining structuralmotif of the family, with homology among the various
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E A A T
Cellular excitatory amino acid transporters
Richard J. Bridges, Professor of PharmacologyCenter for Structural and Functional
Neuroscience, Department of PharmaceuticalSciences, School of Pharmacy and AlliedHealth Sciences, The University of MontanaMissoula, MT 59812-1075, USA.
Richard J. Bridges is a professor at theDepartment of Pharmaceutical Sciences,University of Montana. His research focuses onthe pharmacological specificity and physiologicalroles of glutamate transporters in the brain andspinal cord.
Table 1. Differentiation of the excitatory amino acid transporter
Subtype Primary Distribution Distinguishing Pharmacologicalin Brain Features
EAAT1 Cerebellar glia 4-MG and -SOS as substratesGLAST,
EAAT2 Forebrain glia , , - -2,3-PDC and 3-TMGGLT1, as non-transportable inhibitors
EAAT3 Cortical neurons -aspartate- -hydroxamate as anEAAC1, inhibitor
EAAT4 Cerebellar Purkinje neurons - -AA as a substrate
EAAT5 Retina and as non-transportable inhibitors
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(Bold text denotes compounds available from Tocris)
Abbreviations include: 4-MG = (2 ,4 )-4-methylglutamate; L-SOS = L-serine-O-sulfate; DHK = Dihydrokainic acid; 3-TMG = (±)- -3-
Methylglutamic acid; L- -AA = L- -aminoadipate; THA = - -hydroxy-aspartate
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transporters running at about 40-50%. Assummarized in Table 1, EAAT1-5 exhibit distinctcellular and anatomical distribution, as well as markeddifferences in pharmacological specificity.
The presence of distinct EAAT subtypes naturallyleads to questions regarding the possibility of subtype-specific roles and, consequently, to the needfor selective inhibitors with which to probe function.Toward this goal, the cloning and expression of individual transporters in model systems (especially
oocytes) has not only provided a strategy for selective characterization, but has also allowedtransport to be readily quantified byelectrophysiological methods. As EAAT-mediatedtransport is electrogenic, uptake can be followed bymeasuring substrate-induced currents (see multiplechapters in ). Indeed, substrate activities in thesestudies are typically reported as a percentage of amaximum current (% Imax) generated by knownsubstrate, such as -glutamate, rather than as apercentage of the rate of the accumulation of a [ H]-substrate, such as [ H]- -aspartate or [ H]- -glutamate. This approach has also proven quiteadvantageous in resolving another keypharmacological issue problematic to standard
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radiolabeled substrate-based competition assays; thatis the differentiation of substrate and non-substrateinhibitors (also known as non-transportableinhibitors). For example, the ability of an analog tocompetitively block the uptake of a radiolabeledsubstrate indicates that the analog is likely binding tothe substrate site on the transporter protein, butprovides little or no insight as to whether or not it canalso be translocated intracellularly. Interestingly,recent structure-activity studies on EAAT2 suggestthat the structural motifs regulating binding andsubstrate translocation are not necessarily one andthe same.
As is the case with -glutamate, all of the EAATsshare the ability to utilize -aspartate as a substrate.
Further, the aspartate-based analog, - -hydroxy-aspartate (THA) also competitively blocks uptakethrough all the EAATs, acting as an alternativesubstrate at EAAT1-4 and as a non-transportableinhibitor at EAAT5. The much usedconformationally-restricted glutamate analog, - -2,4-pyrrolidine dicarboxylate ( - -2,4-PDC) has asimilar specificity, acting as a substrate of EAAT1-4
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EAAT pharmacology
Summary of glutamate transport mechanisms
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excitatory signal needs to be evaluated in terms morecomplex than just simple uptake into surroundingcells. Emerging issues of interest include:
) understanding why transport inhibitors alter postsynaptic signaling in only certain subsets of glutamatergic synapses,
) determining the significance of binding to, andtranslocation through, the transporters relative to thelevels and timecourse of glutamate in the synapticcleft and
) elucidating the role of transport as a variableinfluencing the extent of synaptic spillover.
Another area of focus will be the use of novelsubstrates and inhibitors to probe the mechanism andphysiological significance of the chlorideconductances associated with EAATactivity. With respect to the vesicular glutamate transporter, more potent and selectiveinhibitors will be particularly useful in determining therole of the transporter in establishing the vesicular content of -glutamate and, potentially, the amounts of synaptically released transmitter. Significantly, arecent study reported that treating synaptosomes withthe inhibitory dye Rose Bengal resulted in a reduction
in the depolarization-induced release of -glutamate.The potential to modulate excitatory activity in thismanner will certainly attract marked attention in thenear future.
Overall, the availability of an increasing number of pharmacological agents with which to selectivelymodulate the activity of the various glutamatetransporters has been, and will continue to be, acritical step in elucidating their respective roles. Whilethere is still much left to be accomplished, it isrewarding to see how these compounds havesparked a growing interest in glutamate transport andthe recognition that it is an integral part of theexcitatory amino acid system.
The author would like to thank M.P. Kavanaugh, C.M.Thompson and A.R. Chamberlin for their invaluablediscussions and input.
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Acknowledgement
vesicles. Interestingly, the vesicular system has alsobeen shown to be competitively blocked by theglutamate metabotropic receptor agonist -ACPDand ionotropic receptor antagonist kynurenate.Further, the kynurenate analogs xanthurenate and 7-Cl-kynurenate are slightly more effective as inhibitorsthan kynurenate itself. Two of the most potentinhibitor classes identified to date are the ergots, (e.g.bromocriptine ) and the azo dyes, (e.g. Evans Blue,Chicago Sky Blue ). These azo dyes are reported toinhibit the vesicular system with K values three to four
orders of magnitude below the K for -glutamate. Inaddition to these small molecules, recent studieshave also identified a family of inhibitory proteinfactors (IPF , IPF and IPF ) that strongly bind to thetransporter and block glutamate uptake into synapticvesicles. While more systematic structure activitystudies will be needed to develop a detailedpharmacophore model of the substrate site onvesicular glutamate transporters, the inhibitorsdescribed above should prove useful for bothmodulating transporter activity in functional studiesand the design of more specific analogs.
The library of compounds with which to characterizethese various glutamate transport systems ismultiplying rapidly, as is the number of studiesemploying selective substrates and inhibitors asprobes of structure and function. Continued structure-activity analyses with conformationally constrainedanalogs will lead to increasingly detailedpharmacophore models of the substrate bindingdomains that should eventually be incorporated intothe emerging topological models of transporter protein structure. Of particular value in these effortswill be the development of compounds, such asphotoaffinity labels, that can effectively bind to thesubstrate site on the transporters and covalentlymodify the participating amino acids. From a
functional perspective, a more complete library of selective inhibitors and substrates should also help tounravel the role of transport in excitatory signaling.
Accumulating evidence suggests that the potentialrole of the EAATs in shaping the postsynaptic
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Future directions
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0271 -ACBD ..........................................................................Potent, selective -glutamate uptake inhibitor 0332 L-CCG-III...........................................................................Potent, competitive uptake inhibitor 0846 Chicago Sky Blue 6B ........................................................Potent inhibitor of -glutamate uptake into synaptic
vesicles0237 7-Chlorokynurenic acid .....................................................Potent, competitive inhibitor of -glutamate uptake0134 (±)-Chlorpheg.................................................................... -homocysteate uptake inhibitor 0818 (2 ,3 )-Chlorpheg ............................................................ -homocysteate uptake inhibitor 0111 Dihydrokainic acid.............................................................EAAT2(GLT1)-selective non-transportable inhibitor
of -glutamate and -aspartate uptake
0845 Evans Blue........................................................................Potent inhibitor of -glutamate uptake into synapticvesicles0182 D(+)- -3-Hydroxyaspartic acid....................................Potent, competitive, transportable inhibitor 0183 L(-)- -3-Hydroxyaspartic acid .....................................Potent, competitive, transportable EAAT1-4
inhibitor/non-transportable EAAT5 inhibitor 0811 (±)- -3-Methylglutamic acid ........................................EAAT2 blocker 0973 MPDC ...............................................................................Potent inhibitor of -glutamate uptake. Less activity
as a substrate compared to its parent compound(0298)
0298 L- -2,4-PDC................................................................Potent, competitive, transportable EAAT1-4inhibitor/non-transportable EAAT5 inhibitor
1223 -TBOA ...........................................................................Potent, selective non-transportable inhibitor of EAATs
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The Ins and Outs of Glutamate Transporter Pharmacology,Tocris Reviews No. 17, June 2001
©2001 Tocris CooksonPublished and distributed by Tocris Cookson, Bristol, UK
Editors: Samantha Manley, Ph.D., Natalie Barker, B.Sc.Managing Editor: Duncan Crawford, Ph.D.Design and Production: Jane Champness; Lacia Ashman, MA