Download - Glutamate Rev

7/28/2019 Glutamate Rev

http://slidepdf.com/reader/full/glutamate-rev 1/6

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

L

L

L

11,16,34,36,63

13,25,26,42,44,52,55

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

L

L

L

62

+ +

+ 31,62

29,43,50

2-4,22

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

L

L

L

L

rat

trans

rat

rabbit

DHK MPDC

THA - -2,4-PDC

b

a

L trans

(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

S R threo

threoa a b

Tocris Cookson Inc., USAToll Free Tel: (800) 421-3701 Tel: (636) 207-7651

Toll Free Fax: (800) 483-1993 Fax: (636) 207-7683e-mail: [email protected]

Tocris Cookson Ltd., UKTel: + 44 (0)117 982 6551Fax: + 44 (0)117 982 6552e-mail: [email protected] e-mail: [email protected]



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

Xenopus

1

3

3 3

L

D L

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

30

21,30

2,3,22,32

L

D

L

L

b threo

transtrans

EAAT pharmacology

Summary of glutamate transport mechanisms

3 NaH

+

+

3 N a

H +

+

3 NaH

+

+POST-SYNAPTIC NEURON



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.

i

ii

iii

6,17,28,47,54

5,10,17,35,39

9,18,48

2,22,39,40,57,58

38

L

L

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

59

24,59

7

15

46

i

41

trans

m L

a b g

Future directions

References

er 5, a retinal glutamate transporter coupled to a chloride conductance.Proc.Natl.Acad.

Sci.USA 4155.3. (1994) Functional comparisons of three

glutamate transporter subtypes cloned from humanmotor cortex. J.Neurosci 5559.

4. (1993) Cloning and expression of ahuman neutral amino acid transporter withstructural similarity to the glutamate transporter gene family. J.Biol.Chem 15329.

5. (2000) Fast removal of synaptic glutamate by postsynaptic transporters.Neuron 547.

6. (1994) Prolonged presence of glutamate during excitatory synaptic transmissionto cerebellar Purkinje cells. Neuron 1331.

7. (1998) Substituted quinolines as

inhibitors of L-glutamate transport into synapticvesicles. Neuropharmacology 839.

8. (2000) Uptake of glutamate intosynaptic vesicles by an inorganic phosphatetransporter. Science 957.

9. (1999) Clearance of glutamate insidethe synapse and beyond. Curr.Opin.Neurobiol.293.

94

14

268

28

12

37

289

9

Arriza

Arriza

Auger and Attwell

Barbour

Bartlett

Bellocchio

Bergles

et al

.

et al

.

et al

et al

et al

et al

1. Neurotransmitter Transporters. In Meth. Enzymol.Ed. S.G. Amara, Vol 296, Academic Press, SanDiego, 1998.

2. (1997) Excitatory amino acidtransportArriza et al

10. (1997) Synaptic activation of glutamate transporters in hippocampal astrocytes.Neuron 1297.

11. (1998) Neuropharmacologyof AMPA and kainate receptors.Neuropharmacology 1187.

12. (2000) A new structuralclass of subtype-selective inhibitor of clonedexcitatory amino acid transporter, EAAT2. Eur.J.Pharmacol 41.

13. (1999) A pharmacological review of competitive inhibitors and substrates of high-affinity, sodium-dependent glutamate transportersin the central nervous system. Curr.Pharmaceut.Des. 363.

14. (1994) A conformationallyconstrained competitive inhibitor of the sodium-dependent glutamate transporter in forebrainsynaptosomes: 1-anti-endo-3,4-methanopyrrolidinedicarboxylate. Neurosci.Lett. 193.

15. (1989) Glutamate uptake intosynaptic vesicles: Competitive inhibition bybromocriptine. J.Neurochem 1889.

16. (1995) Calcium: still center-stage in hypoxic-ischemic neuronal death. TiNS 58.

17. (1997) Transporters buffer synaptically released glutamate on a millisecondtime scale. J.Neurosci 4672.

18. (2000) Synaptically releasedglutamate does not overwhelm transporters on

Bergles and Jahr

Bleakman and Lodge

Brauner-Osborne

Bridges

Bridges

Carlson

Choi

Diamond and Jahr

Diamond and Jahr

19

37

406

5

174

53

18

17

et al

.

et al

et al

et al

.

.



and initial characterization. Proc.Natl.Acad.Sci.USA 4137.

42. (1998) Glutamate transport andstorage in synaptic vesicles. Jap.J.Pharmacol.1.

43. (1992) Cloning and expression of a ratbrain L-glutamate transporter. Nature 464.

44. (1998) The family of sodium-dependentglutamate transporters: a focus on the GLT-1/EAAT2 subtype. Neurochem.Int. 479.

45. (1993) Inhibition of glutamateuptake with L-trans-pyrrolidine-2,4-dicarboxylate

potentiates glutamate toxicity in primaryhippocampal cultures. J.Neurochem. 586.

46. (1995) Uptake of L-glutamate into ratbrain synaptic vesicles: effect of inhibitors that bindspecifically to the glutamate transporter.J.Neurochem. 96.

47. (1993) Glutamate uptake from thesynaptic cleft does not shape the decay of the non-NMDA component of the synaptic current. Neuron

541.48. (1997) Use-dependent increases in

glutamate concentration activate presynapticmetabotropic glutamate receptors. Nature 630.

49. (1998) DL-Threo- -benzyloxyaspartate, a potent blocker of excitatory

amino acid transporters. Mol.Pharmacol. 195.50. (1992) Structure, expression, and

functional analysis of a Na -dependentglutamate/aspartate transporter from rat brain.Proc.Natl.Acad.Sci.USA 10955.

51. (1992) Glutamate transport into synapticvesicles: roles of membrane potential, pH gradient,and intravesicular pH. J.Biol.Chem. 15412.

52. (1997) The role of glutamatetransporters in glutamate homeostasis in the brain.J.Exp.Biol. 401.

53. (2000) Identification of a vesicular glutamate transporter that defines a glutamergicphenotype in neurons. Nature 189.

54. (1994) Block of glutamate

transporters potentiates postsynaptic excitation.Neuron 1195.

55. (1998) Molecular pharmacology andphysiology of glutamate transporters in the centralnervous system. Clin.Exp.Pharmacol.Physiol.393.

56. (1997) Contrasting modes of action of methylglutamate derivatives on theexcitatory amino acid transporters, EAAT1 andEAAT2. Mol.Pharmacol. 809.

57. (1995) Ion fluxes associated withexcitatory amino acid transport. Neuron 721.

58. (1998) Macroscopicand microscopic properties of a cloned glutamatetransporter/chloride channel. J.Neurosci. 7650.

59. (1993) Glutamate uptake systemin the presynaptic vesicle: glutamic acid analogs asinhibitors and alternate substrates. Neurochemical.Res. 79.

60. (1996) Regulation of glutamatetransport into synaptic vesicles by chloride andproton gradient. J.Biol.Chem. 11726.

61. (1995) Inhibition by folded isomersof 1-2-(carboxycyclopropyl)glycine of glutamateuptake via the human glutamate transporter hGluT-1. Eur.J.Pharmacol. 387.

62. (1996) Flux coupling ina neuronal glutamate transporter. Nature 634.

63. (1999) Recent excitement in the ionotropicglutamate receptor field. Ann.N.Y.Acad.Sci.

465.

94

77

360

33

61

65

11

385

53

89

267

200

407

13

25

51

15

18

18

271

289

383

868

Ozkan and Ueda

Pines

Robinson

Robinson

Roseth

Sarantis

Scanziani

Shimamoto

Storck

Tabb

Takahashi

Takamori

Tong and Jahr

Vandenberg

Vandenberg

Wadiche

Wadiche and Kavanaugh

Winter and Ueda

Wolosker

Yamashita

Zerangue and Kavanaugh

Ziff

et al

et al

et al

et al

et al

et al

et al

et al

et al

et al

et al

et al

et al

et al

b

+

hippocampal astrocytes during high-frequencystimulation. J.Neurophysiol. 2835.

19. (1996) Comparison of Na -dependentglutamate transport activity in synaptosomes, C6glioma, and Xenopus oocytes expressingexcitatory amino acid carrier 1 (EAAC1). Mol.Pharmacol. 465.

20. (2001) Pharmacologicalcharacterization of threo-3-methyglutamic acid withexcitatory amino acid transporters in native andrecombinant systems. J.Neurochem 109.

21. (1998) Structural determinants of

substrates and inhibitors: probing glutamatetransporters with meso-2,4-methano-2,4-pyrrolidine dicarboxylate. Bioorg.Med.Chem.Lett3101.

22. (1995) An excitatory amino acidtransporter with properties of a ligand-gatedchloride channel. Nature 599.

23. (1996) Amino acidneurotransmission: dynamics of vesicular uptake.Neurochemical.Res 1053.

24. (1992) Inhibit ion of L-glutamate intosynaptic vesicles. Neurosci.Lett. 125.

25. (1997) Cellular distribution and kinetic properties of the high-affinity glutamate transporters. Brain Res.Bull

233.26. (1997) High affinity

glutamate transporters: regulation of expressionand activity. Mol.Pharmacol. 6.

27. (1993) An anion binding sitethat regulates the glutamate transporter of synapticvesicles. J.Biol.Chem. 23122.

28. (1993) The uptake inhibitor L-trans-PDC enhances responses to glutamate butfails to alter kinetics of excitatory synaptic currentsin the hippocampus. J.Neurophysiol 2187.

29. (1992) Primary structure andfunctional characterization of a high-affinityglutamate transporter. Nature 467.

30. (1999) Differentiation of substrate and

nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters. Mol.Pharmacol.

1095.31. (1998) Stoichiometry of the glial

glutamate transporter GLT-1 expressed inducibly ina chinese hamster ovary cell line selected for lowendogenous Na+-dependent glutamate uptake.J.Neurosci 9620.

32. (1998) Molecular cloning and expressionof the rat EAAT4 glutamate transporter subtype.Mol.Brain Res. 174.

33. (1990) Amino acidneurotransmission: spotlight on synaptic vesicles.TiNS 83.

34. (2000) Glutamate as a neurotransmitter

in the brain: review of physiology and pathology.J.Nutr 1007S.35. (1999) Substrate turnover by

transporters curtails synaptic glutamate transients.J.Neurosci. 9242.

36. (1998) Molecular determinants of NMDA receptor pharmacological diversity. Prog.Brain Res. 171.

37. (1985) Characterization of glutamate uptake into synaptic vesicles.J.Neurochem 99.

38. (2001) Inhibition of vesicular glutamatestorage and exocytotic release by Rose Bengal.J.Neurochem. 34.

39. (1998) Anion currents and predictedglutamate flux through a neuronal glutamatetransporter. J.Neurosci. 7099.

40. (2000) Isolation of currentcomponents and partial reaction cycles in the glialglutamate transporter EAAT2. J.Neurosci. 2749.

41. (1997) A protein factor that inhibits ATP-dependent glutamate and GABAaccumulation into synaptic vesicles: purification

83

49

76

8

375

21

135

45

52

268

70

360

56

18

63

13

130

19

116

44

77

18

20

Dowd

Eliasof

Esslinger

Fairman

Fykse and Fonnum

Fykse

Gegelashvili and Schousboe

Gegelashvili and Schousboe

Hartinger and Jahn

Isaacson and Nicoll

Kanai and Hediger

Koch

Levy

Lin

Maycox

Meldrum

Mennerick

Monaghan

Naito and Ueda

Ogita

Otis and Jahr

Otis and Kavanaugh

Ozkan

et al

et al

.

et al

.

et al

.

et al

.

.

et al

et al

.

et al

et al

.

et al

et al

.

et al

et al

+


http://slidepdf.com/reader/full/glutamate-rev 6/[email protected]

Tocris Cookson Inc.16144 Westwoods Business Park

Ellisville MO 63021 USAToll Free Tel: (800) 421-3701 Tel: (636) 207-7651

Toll Free Fax: (800) 483-1993 Fax: (636) [email protected]

EAA(0601)

Tocris Cookson Ltd.Northpoint Fourth WayAvonmouth Bristol BS11 8TA UKTel: + 44 (0)117 982 6551Fax: + 44 (0)117 982 [email protected]

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

cis

S R

threothreo

threo

trans

L

L

L

L

L

L L

L

L

DL

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

Download - Glutamate Rev

Top Related