Dance Lessons for Proteins: The Dynamics and Thermodynamics of a Sodium/Aspartate Symporter

Part of the Springer Series in Biophysics book series (BIOPHYSICS, volume 17)


Secondary active transporters harvest the energy of the ionic gradients to drive concentrative uptake of their substrates. This process entails a series of protein conformational transitions that are coupled to binding and unbinding of ions and substrates on the extracellular and intracellular sides of the membrane. Over the last decade, crystallography has provided a growing number of structural snapshots of the transport cycle for several ion-driven transporters. Already these structures, although intrinsically static, have revealed a remarkable plasticity encoded in the architecture of these proteins. Because internal dynamics is an essential feature of transporters, it is necessary to complement crystallographic studies with other techniques that provide information on the ensemble properties of these proteins as well as on the conformational dynamics of individual molecules. Here, we will discuss the emerging approaches to obtain thermodynamic and dynamic information on transporters using a sodium/aspartate symporter from Pyrococcus horikoshii, GltPh, as a model system. GltPh is a bacterial homologue of the mammalian glutamate transporters, for which crystal structures of several states have been determined, providing a framework for further mechanistic studies. We will discuss how within this system the equilibrium and kinetic studies based on the isothermal titration calorimetry, fluorescence, and electron paramagnetic resonance spectroscopy inform us on the energetic relationship between the key functional states, and mechanisms of coupling between transport cycle and ionic gradients. We will further describe how single molecule studies open doors to a detailed characterization of the timing and order of the conformational transitions underlying transport processes.


Glutamate transporters Sodium symporters Functional dynamics Anion permeation Substrate binding Alternating access Secondary transport Conformational changes 


  1. Akyuz N, Altman R et al (2013) Transport dynamics of a glutamate transporter homologue. Nature 502(7469):114–118PubMedCrossRefGoogle Scholar
  2. Andersen OS, Koeppe RE 2nd (2007) Bilayer thickness and membrane protein function: an energetic perspective. Annu Rev Biophys Biomol Struct 36:107–130PubMedCrossRefGoogle Scholar
  3. Arriza JL, Eliasof S et al (1997) Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U S A 94(8):4155–4160PubMedCrossRefPubMedCentralGoogle Scholar
  4. Barbour B, Brew H et al (1988) Electrogenic glutamate uptake in glial cells is activated by intracellular potassium. Nature 335(6189):433–435PubMedCrossRefGoogle Scholar
  5. Bastug T, Heinzelmann G et al (2012) Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1. PLoS One 7(3):e33058PubMedCrossRefPubMedCentralGoogle Scholar
  6. Bendahan A, Armon A et al (2000) Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter. J Biol Chem 275(48):37436–37442PubMedCrossRefGoogle Scholar
  7. Borre L, Kavanaugh MP et al (2002) Dynamic equilibrium between coupled and uncoupled modes of a neuronal glutamate transporter. J Biol Chem 277(16):13501–13507PubMedCrossRefGoogle Scholar
  8. Boudker O, Verdon G (2010) Structural perspectives on secondary active transporters. Trends Pharmacol Sci 31(9):418–426PubMedCrossRefPubMedCentralGoogle Scholar
  9. Boudker O, Ryan RM et al (2007) Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445(7126):387–393PubMedCrossRefGoogle Scholar
  10. Bouvier M, Szatkowski M et al (1992) The glial cell glutamate uptake carrier countertransports pH-changing anions. Nature 360(6403):471–474PubMedCrossRefGoogle Scholar
  11. Choi DW (1994) Calcium and excitotoxic neuronal injury. Ann N Y Acad Sci 747:162–171PubMedCrossRefGoogle Scholar
  12. Claxton DP, Quick M et al (2010) Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters. Nat Struct Mol Biol 17(7):822–829PubMedCrossRefPubMedCentralGoogle Scholar
  13. Crisman TJ, Qu S et al (2009) Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats. Proc Natl Acad Sci U S A 106(49):20752–20757PubMedCrossRefPubMedCentralGoogle Scholar
  14. Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65(1):1–105PubMedCrossRefGoogle Scholar
  15. DeChancie J, Shrivastava IH et al (2010) The mechanism of substrate release by the aspartate transporter GltPh: insights from simulations. Mol Biosyst 7(3):832–842PubMedCrossRefPubMedCentralGoogle Scholar
  16. Eliasof S, Jahr CE (1996) Retinal glial cell glutamate transporter is coupled to an anionic conductance. Proc Natl Acad Sci U S A 93(9):4153–4158PubMedCrossRefPubMedCentralGoogle Scholar
  17. Ewers D, Becher T et al (2013) Induced fit substrate binding to an archeal glutamate transporter homologue. Proc Natl Acad Sci U S A 110(30):12486–12491PubMedCrossRefPubMedCentralGoogle Scholar
  18. Faham S, Watanabe A et al (2008) The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321(5890):810–814PubMedCrossRefPubMedCentralGoogle Scholar
  19. Fairman WA, Vandenberg RJ et al (1995) An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature 375(6532):599–603PubMedCrossRefGoogle Scholar
  20. Fischbarg J (1988) On the possible permeation of water across the glucose transporter. Mol Cell Biochem 82(1–2):107–111PubMedGoogle Scholar
  21. Focke PJ, Moenne-Loccoz P et al (2011) Opposite movement of the external gate of a glutamate transporter homolog upon binding cotransported sodium compared with substrate. J Neurosci 31(16):6255–6262PubMedCrossRefPubMedCentralGoogle Scholar
  22. Forrest LR, Rudnick G (2009) The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters. Physiology (Bethesda) 24:377–386CrossRefGoogle Scholar
  23. Gendreau S, Voswinkel S et al (2004) A trimeric quaternary structure is conserved in bacterial and human glutamate transporters. J Biol Chem 279(38):39505–39512PubMedCrossRefGoogle Scholar
  24. Georgieva ER, Borbat PP et al (2013) Conformational ensemble of the sodium-coupled aspartate transporter. Nat Struct Mol Biol 20(2):215–221PubMedCrossRefPubMedCentralGoogle Scholar
  25. Grewer C, Balani P et al (2005) Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other. Biochemistry 44(35):11913–11923PubMedCrossRefPubMedCentralGoogle Scholar
  26. Groeneveld M, Slotboom DJ (2007) Rigidity of the subunit interfaces of the trimeric glutamate transporter GltT during translocation. J Mol Biol 372(3):565–570PubMedCrossRefGoogle Scholar
  27. Groeneveld M, Slotboom DJ (2010) Na(+):aspartate coupling stoichiometry in the glutamate transporter homologue Glt(Ph). Biochemistry 49(17):3511–3513PubMedCrossRefGoogle Scholar
  28. Grunewald M, Bendahan A et al (1998) Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology. Neuron 21(3):623–632PubMedCrossRefGoogle Scholar
  29. Hanelt I, Wunnicke D et al (2013) Conformational heterogeneity of the aspartate transporter Glt(Ph). Nat Struct Mol Biol 20(2):210–214PubMedCrossRefGoogle Scholar
  30. Holley DC, Kavanaugh MP (2009) Interactions of alkali cations with glutamate transporters. Philos Trans R Soc Lond B Biol Sci 364(1514):155–161PubMedCrossRefPubMedCentralGoogle Scholar
  31. Huang Z, Tajkhorshid E (2008) Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter. Biophys J 95(5):2292–2300PubMedCrossRefPubMedCentralGoogle Scholar
  32. Huang Z, Tajkhorshid E (2010) Identification of the third Na+ site and the sequence of extracellular binding events in the glutamate transporter. Biophys J 99(5):1416–1425PubMedCrossRefPubMedCentralGoogle Scholar
  33. Jardetzky O (1966) Simple allosteric model for membrane pumps. Nature 211(5052):969–70PubMedCrossRefGoogle Scholar
  34. Kanner BI, Sharon I (1978) Active transport of l-glutamate by membrane vesicles isolated from rat brain. Biochemistry 17(19):3949–3953PubMedCrossRefGoogle Scholar
  35. Kavanaugh MP (1998) Neurotransmitter transport: models in flux. Proc Natl Acad Sci U S A 95(22):12737–12738PubMedCrossRefPubMedCentralGoogle Scholar
  36. Koch HP, Larsson HP (2005) Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters. J Neurosci 25(7):1730–1736PubMedCrossRefGoogle Scholar
  37. Koch HP, Hubbard JM et al (2007) Voltage-independent sodium-binding events reported by the 4B–4C loop in the human glutamate transporter excitatory amino acid transporter 3. J Biol Chem 282(34):24547–24553PubMedCrossRefGoogle Scholar
  38. Krishnamurthy H, Piscitelli CL et al (2009) Unlocking the molecular secrets of sodium-coupled transporters. Nature 459(7245):347–355PubMedCrossRefGoogle Scholar
  39. Larsson HP, Wang X et al (2010) Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model. Proc Natl Acad Sci U S A 107(31):13912–13917PubMedCrossRefPubMedCentralGoogle Scholar
  40. Lau A, Tymianski M (2010) Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch 460(2):525–542PubMedCrossRefGoogle Scholar
  41. Leary GP, Stone EF et al (2007) The glutamate and chloride permeation pathways are colocalized in individual neuronal glutamate transporter subunits. J Neurosci 27(11):2938–2942PubMedCrossRefGoogle Scholar
  42. Lee SG, Su ZZ et al (2008) Mechanism of ceftriaxone induction of excitatory amino acid transporter-2 expression and glutamate uptake in primary human astrocytes. J Biol Chem 283(19):13116–13123PubMedCrossRefPubMedCentralGoogle Scholar
  43. Li J, Shaikh SA et al (2013) Transient formation of water-conducting states in membrane transporters. Proc Natl Acad Sci U S A 110:7696–7701PubMedCrossRefPubMedCentralGoogle Scholar
  44. Majumdar DS, Smirnova I et al (2007) Single-molecule FRET reveals sugar-induced conformational dynamics in LacY. Proc Natl Acad Sci U S A 104(31):12640–12645PubMedCrossRefPubMedCentralGoogle Scholar
  45. Nelson PJ, Dean GE et al (1983) Hydrogen ion cotransport by the renal brush border glutamate transporter. Biochemistry 22(23):5459–5463PubMedCrossRefGoogle Scholar
  46. Oldham ML, Chen J (2011) Crystal structure of the maltose transporter in a pretranslocation intermediate state. Science 332(6034):1202–1205PubMedCrossRefGoogle Scholar
  47. Perez C, Koshy C et al (2012) Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP. Nature 490(7418):126–130PubMedCrossRefGoogle Scholar
  48. Picaud SA, Larsson HP et al (1995) Glutamate-gated chloride channel with glutamate-transporter-like properties in cone photoreceptors of the tiger salamander. J Neurophysiol 74(4):1760–1771PubMedGoogle Scholar
  49. Ressl S, Terwisscha van Scheltinga AC et al (2009) Molecular basis of transport and regulation in the Na(+)/betaine symporter BetP. Nature 458(7234):47–52PubMedCrossRefGoogle Scholar
  50. Reyes N, Ginter C et al (2009) Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462(7275):880–885PubMedCrossRefPubMedCentralGoogle Scholar
  51. Reyes N, Oh S et al (2013) Binding thermodynamics of a glutamate transporter homologue. Nat Struct Mol Biol 20(5):634–640PubMedCrossRefPubMedCentralGoogle Scholar
  52. Rosental N, Gameiro A et al (2011) A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters. J Biol Chem 286(48):41381–41390PubMedCrossRefPubMedCentralGoogle Scholar
  53. Rothstein JD, Dykes-Hoberg M et al (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16(3):675–686PubMedCrossRefGoogle Scholar
  54. Rothstein JD, Patel S et al (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433(7021):73–77PubMedCrossRefGoogle Scholar
  55. Ryan RM, Mindell JA (2007) The uncoupled chloride conductance of a bacterial glutamate transporter homolog. Nat Struct Mol Biol 14(5):365–371PubMedCrossRefGoogle Scholar
  56. Ryan RM, Vandenberg RJ (2002) Distinct conformational states mediate the transport and anion channel properties of the glutamate transporter EAAT-1. J Biol Chem 277(16):13494–13500PubMedCrossRefGoogle Scholar
  57. Ryan RM, Mitrovic AD et al (2004) The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway. J Biol Chem 279(20):20742–20751PubMedCrossRefGoogle Scholar
  58. Ryan RM, Compton EL et al (2009) Functional characterization of a Na+-dependent aspartate transporter from Pyrococcus horikoshii. J Biol Chem 284(26):17540–17548PubMedCrossRefPubMedCentralGoogle Scholar
  59. Schiemann O, Weber A et al (2003) Nanometer distance measurements on RNA using PELDOR. J Am Chem Soc 125(12):3434–3435PubMedCrossRefGoogle Scholar
  60. Seal RP, Leighton BH et al (1998) Transmembrane topology mapping using biotin-containing sulfhydryl reagents. Methods Enzymol 296:318–331PubMedCrossRefGoogle Scholar
  61. Seal RP, Shigeri Y et al (2001) Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance. Proc Natl Acad Sci U S A 98(26):15324–15329PubMedCrossRefPubMedCentralGoogle Scholar
  62. Shimamura T, Weyand S et al (2010) Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328(5977):470–473PubMedCrossRefPubMedCentralGoogle Scholar
  63. Shlaifer I, Kanner BI (2007) Conformationally sensitive reactivity to permeant sulfhydryl reagents of cysteine residues engineered into helical hairpin 1 of the glutamate transporter GLT-1. Mol Pharmacol 71(5):1341–1348PubMedCrossRefGoogle Scholar
  64. Shrivastava IH, Jiang J et al (2008) Time-resolved mechanism of extracellular gate opening and substrate binding in a glutamate transporter. J Biol Chem 283(42):28680–28690PubMedCrossRefPubMedCentralGoogle Scholar
  65. Slotboom DJ, Sobczak I et al (1999) A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop. Proc Natl Acad Sci U S A 96(25):14282–14287PubMedCrossRefPubMedCentralGoogle Scholar
  66. Stolzenberg S, Khelashvili G et al (2012) Structural intermediates in a model of the substrate translocation path of the bacterial glutamate transporter homologue GltPh. J Phys Chem B 116(18):5372–5383PubMedCrossRefPubMedCentralGoogle Scholar
  67. Tao Z, Gameiro A et al (2008) Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1. Biochemistry 47(48):12923–12930PubMedCrossRefPubMedCentralGoogle Scholar
  68. Tao Z, Rosental N et al (2010) Mechanism of cation binding to the glutamate transporter EAAC1 probed with mutation of the conserved amino acid residue Thr101. J Biol Chem 285(23):17725–17733PubMedCrossRefPubMedCentralGoogle Scholar
  69. Teichman S, Kanner BI (2007) Aspartate-444 is essential for productive substrate interactions in a neuronal glutamate transporter. J Gen Physiol 129(6):527–539PubMedCrossRefPubMedCentralGoogle Scholar
  70. Teichman S, Qu S et al (2012) Conserved asparagine residue located in binding pocket controls cation selectivity and substrate interactions in neuronal glutamate transporter. J Biol Chem 287(21):17198–17205PubMedCrossRefPubMedCentralGoogle Scholar
  71. Vandenberg RJ, Handford CA et al (2011) Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1. Biochem J 439(2):333–340PubMedCrossRefGoogle Scholar
  72. Verdon G, Boudker O (2012) Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog. Nat Struct Mol Biol 19(3):355–357PubMedCrossRefPubMedCentralGoogle Scholar
  73. Veruki ML, Morkve SH et al (2006) Activation of a presynaptic glutamate transporter regulates synaptic transmission through electrical signaling. Nat Neurosci 9(11):1388–1396PubMedCrossRefGoogle Scholar
  74. Wadiche JI, Amara SG et al (1995a) Ion fluxes associated with excitatory amino acid transport. Neuron 15(3):721–728PubMedCrossRefGoogle Scholar
  75. Wadiche JI, Arriza JL et al (1995b) Kinetics of a human glutamate transporter. Neuron 14(5):1019–1027PubMedCrossRefGoogle Scholar
  76. Watzke N, Bamberg E et al (2001) Early intermediates in the transport cycle of the neuronal excitatory amino acid carrier EAAC1. J Gen Physiol 117(6):547–562PubMedCrossRefPubMedCentralGoogle Scholar
  77. Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283(5408):1676–1683PubMedCrossRefGoogle Scholar
  78. Weyand S, Shimamura T et al (2008) Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322(5902):709–713PubMedCrossRefPubMedCentralGoogle Scholar
  79. Yamashita A, Singh SK et al (2005) Crystal structure of a bacterial homologue of Na+/Cl–dependent neurotransmitter transporters. Nature 437(7056):215–223PubMedCrossRefGoogle Scholar
  80. Yernool D, Boudker O et al (2003) Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42(44):12981–12988PubMedCrossRefGoogle Scholar
  81. Yernool D, Boudker O et al (2004) Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431(7010):811–818PubMedCrossRefGoogle Scholar
  82. Yi JH, Hazell AS (2006) Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury. Neurochem Int 48(5):394–403PubMedCrossRefGoogle Scholar
  83. Yin Y, He X et al (2006) Structure of the multidrug transporter EmrD from Escherichia coli. Science 312(5774):741–744PubMedCrossRefPubMedCentralGoogle Scholar
  84. Zerangue N, Kavanaugh MP (1996) Flux coupling in a neuronal glutamate transporter. Nature 383(6601):634–637PubMedCrossRefGoogle Scholar
  85. Zhang Z, Grewer C (2007) The sodium-coupled neutral amino acid transporter SNAT2 mediates an anion leak conductance that is differentially inhibited by transported substrates. Biophys J 92(7):2621–2632PubMedCrossRefPubMedCentralGoogle Scholar
  86. Zhang Z, Tao Z et al (2007) Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1. Proc Natl Acad Sci U S A 104(46):18025–18030PubMedCrossRefPubMedCentralGoogle Scholar
  87. Zhao Y, Terry D et al (2010) Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465(7295):188–193PubMedCrossRefPubMedCentralGoogle Scholar
  88. Zhao Y, Terry DS et al (2011) Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474(7349):109–113PubMedCrossRefPubMedCentralGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Weill Cornell Medical CollegeNew YorkUSA

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