Advertisement

Tracer Flux Measurements to Study Outward Transport by Monoamine Neurotransmitter Transporters

  • Thomas Steinkellner
  • Felix P. Mayer
  • Tina Hofmaier
  • Marion Holy
  • Therese Montgomery
  • Birgit Eisenrauch
  • Michael Freissmuth
  • Harald H. SitteEmail author
Protocol
Part of the Neuromethods book series (NM, volume 118)

Abstract

The physiological role of neurotransmitter transporter (NTT) proteins is the reuptake of released neurotransmitter from the synaptic cleft. NTTs accomplish uptake by undergoing a transport cycle, which relies on a return step in the empty state. In addition, NTTs can also run in the reverse direction and transport substrates out of the cells. This can be observed under conditions, where the transmembrane sodium gradient dissipates, e.g., if sodium accumulates within the cell. This reverse transport mode is also induced by amphetamines and the exact mechanism underlying the amphetamine action is still enigmatic and involves complex regulatory processes. In the current chapter, we describe various methods that can be used to assess the efflux of neurotransmitter from cells heterologously expressing the NTTs of interest or from preparations derived from intact brain tissue.

Key words

Carrier-mediated efflux Transport reversal Neurotransmitter transporter Superfusion Radiolabeled tracer flux Heterologous cell expression systems Synaptosomes Brain slices 

Notes

Acknowledgements

The authors wish to thank the Austrian Science Fund for continuous support (grant F35).

References

  1. 1.
    Iversen LL (1971) Role of transmitter uptake mechanisms in synaptic neurotransmission. Br J Pharmacol 41:571–591CrossRefPubMedGoogle Scholar
  2. 2.
    Rudnick G, Clark J (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters. Biochim Biophys Acta 1144:249–263CrossRefPubMedGoogle Scholar
  3. 3.
    Kristensen AS, Andersen J, Jorgensen TN et al (2011) SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev 63:585–640CrossRefPubMedGoogle Scholar
  4. 4.
    Nelson N (1998) The family of Na+/Cl− neurotransmitter transporters. J Neurochem 71:1785–1803CrossRefPubMedGoogle Scholar
  5. 5.
    Iversen L (2000) Neurotransmitter transporters: fruitful targets for CNS drug discovery. Mol Psychiatry 5:357–362CrossRefPubMedGoogle Scholar
  6. 6.
    Axelrod J, Whitby LG, Hertting G (1961) Effect of psychotropic drugs on the uptake of 3 H-Norepinephrine by tissues. Science 133:383–384CrossRefPubMedGoogle Scholar
  7. 7.
    Jardetzky O (1966) Simple allosteric model for membrane pumps. Nature 211:969–970CrossRefPubMedGoogle Scholar
  8. 8.
    Singh SK, Piscitelli CL, Yamashita A et al (2008) A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322:1655–1661CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Singh SK, Yamashita A, Gouaux E (2007) Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448:952–956CrossRefPubMedGoogle Scholar
  10. 10.
    Yamashita A, Singh SK, Kawate T et al (2005) Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437:215–223CrossRefPubMedGoogle Scholar
  11. 11.
    Forrest LR, Rudnick G (2009) The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters. Physiology (Bethesda) 24:377–386CrossRefGoogle Scholar
  12. 12.
    Forrest LR, Zhang YW, Jacobs MT et al (2008) Mechanism for alternating access in neurotransmitter transporters. Proc Natl Acad Sci U S A 105:10338–10343CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Penmatsa A, Gouaux E (2014) How LeuT shapes our understanding of the mechanisms of sodium-coupled neurotransmitter transporters. J Physiol 592:863–869CrossRefPubMedGoogle Scholar
  14. 14.
    Shi L, Quick M, Zhao Y et al (2008) The mechanism of a neurotransmitter:sodium symporter—inward release of Na+ and substrate is triggered by substrate in a second binding site. Mol Cell 30:667–677CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhao Y, Terry D, Shi L et al (2010) Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465:188–193CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zhao Y, Terry DS, Shi L et al (2011) Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474:109–113CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Barger G, Dale HH (1910) Chemical structure and sympathomimetic action of amines. J Physiol 41:19–59CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Tainter ML, Chang DK (1927) The antagonism of sympathetic and adrenaline content of the spleen, kidney, and salivary glands in the sheep. J Pharmacol Exp Ther 30:193–207Google Scholar
  19. 19.
    Furchgott RF, Kirpekar SM, Rieker M et al (1963) Actions and interactions of norepinephrine, tyramine and cocaine on aortic strips of rabbit and left atria of guinea pig and cat. J Pharmacol Exp Ther 142:39–58PubMedGoogle Scholar
  20. 20.
    Ross SB, Kelder D (1977) Efflux of 5-hydroxytryptamine from synaptosomes of rat cerebral cortex. Acta Physiol Scand 99:27–36CrossRefPubMedGoogle Scholar
  21. 21.
    Glowinski J, Axelrod J (1965) Effect of drugs on the uptake, release, and metabolism of H3-norepinephrine in the rat brain. J Pharmacol Exp Ther 149:43–49PubMedGoogle Scholar
  22. 22.
    Agneter E, Sitte HH, Stockl-Hiesleitner S et al (1995) Sustained dopamine release induced by secretoneurin in the striatum of the rat: a microdialysis study. J Neurochem 65:622–625CrossRefPubMedGoogle Scholar
  23. 23.
    Gainetdinov RR, Fumagalli F, Jones SR et al (1997) Dopamine transporter is required for in vivo MPTP neurotoxicity: evidence from mice lacking the transporter. J Neurochem 69:1322–1325CrossRefPubMedGoogle Scholar
  24. 24.
    Gainetdinov RR, Jones SR, Fumagalli F et al (1998) Re-evaluation of the role of the dopamine transporter in dopamine system homeostasis. Brain Res Brain Res Rev 26:148–153CrossRefPubMedGoogle Scholar
  25. 25.
    Daws LC, Toney GM, Davis DJ et al (1997) In vivo chronoamperometric measurements of the clearance of exogenously applied serotonin in the rat dentate gyrus. J Neurosci Methods 78:139–150CrossRefPubMedGoogle Scholar
  26. 26.
    Gobbi M, Frittoli E, Mennini T et al (1992) Releasing activities of d-fenfluramine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin. Naunyn Schmiedebergs Arch Pharmacol 345:1–6CrossRefPubMedGoogle Scholar
  27. 27.
    Gobbi M, Funicello M, Gerstbrein K et al (2008) N,N-Dimethyl-thioamphetamine and methyl-thioamphetamine, two non-neurotoxic substrates of 5-HT transporters, have scant in vitro efficacy for the induction of transporter-mediated 5-HT release and currents. J Neurochem 105:1770–1780CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Gobbi M, Mennini T, Garattini S (1997) Mechanism of neurotransmitter release induced by amphetamine derivatives: pharmacological and toxicological aspects. Curr Top Pharmacol 3:217–227Google Scholar
  29. 29.
    Rothman RB, Baumann MH (2002) Serotonin releasing agents. Neurochemical, therapeutic and adverse effects. Pharmacol Biochem Behav 71:825–836CrossRefPubMedGoogle Scholar
  30. 30.
    Rothman RB, Baumann MH (2003) Monoamine transporters and psychostimulant drugs. Eur J Pharmacol 479:23–40CrossRefPubMedGoogle Scholar
  31. 31.
    Whittaker VP, Michaelson IA, Kirkland RJ (1964) The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). Biochem J 90:293–303CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Scholze P, Norregaard L, Singer E et al (2002) The role of zinc ions in reverse transport mediated by monoamine transporters. J Biol Chem 277:21505–21513CrossRefPubMedGoogle Scholar
  33. 33.
    Eshleman AJ, Henningsen RA, Neve KA et al (1994) Release of dopamine via the human transporter. Mol Pharmacol 45:312–316PubMedGoogle Scholar
  34. 34.
    Wall SC, Gu H, Rudnick G (1995) Biogenic amine flux mediated by cloned transporters stably expressed in cultured cell lines: amphetamine specificity for inhibition and efflux. Mol Pharmacol 47:544–550Google Scholar
  35. 35.
    Pifl C, Agneter E, Drobny H et al (1999) Amphetamine reverses or blocks the operation of the human noradrenaline transporter depending on its concentration: superfusion studies on transfected cells. Neuropharmacology 38:157–165CrossRefPubMedGoogle Scholar
  36. 36.
    Pifl C, Drobny H, Reither H et al (1995) Mechanism of the dopamine-releasing actions of amphetamine and cocaine: plasmalemmal dopamine transporter versus vesicular monoamine transporter. Mol Pharmacol 47:368–373PubMedGoogle Scholar
  37. 37.
    Pifl C, Singer EA (1999) Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion. Mol Pharmacol 56:1047–1054PubMedGoogle Scholar
  38. 38.
    Seidel S, Singer E, Just H et al (2005) Amphetamines take two to tango: an oligomer-based counter-transport model of neurotransmitter transport explores the amphetamine action. Mol Pharmacol 67:140–151PubMedGoogle Scholar
  39. 39.
    Fog JU, Khoshbouei H, Holy M et al (2006) Calmodulin kinase ii interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport. Neuron 51:417–429CrossRefPubMedGoogle Scholar
  40. 40.
    Steinkellner T, Montgomery TR, Hofmaier T et al (2015) Amphetamine action at the cocaine- and antidepressant-sensitive serotonin transporter is modulated by alphaCaMKII. J Neurosci 35:8258–8271CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Steinkellner T, Mus L, Eisenrauch B et al (2014) In vivo amphetamine action is contingent on alphaCaMKII. Neuropsychopharmacology 39:2681–2693PubMedPubMedCentralGoogle Scholar
  42. 42.
    Steinkellner T, Yang JW, Montgomery TR et al (2012) Ca(2+)/calmodulin-dependent protein kinase IIalpha (alphaCaMKII) controls the activity of the dopamine transporter: implications for Angelman syndrome. J Biol Chem 287:29627–29635CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Rickhag M, Owens WA, Winkler M-T et al (2013) Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release. J Biol Chem 288:27534–27544CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Melikian HE, Buckley KM (1999) Membrane trafficking regulates the activity of the human dopamine transporter. J Neurosci 19:7699–7710PubMedGoogle Scholar
  45. 45.
    Pifl C, Wolf A, Rebernik P et al (2009) Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner. Neuropharmacology 56:531–540CrossRefPubMedGoogle Scholar
  46. 46.
    Foster JD, Yang J-W, Moritz AE et al (2012) Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux. J Biol Chem 287:29702–29712CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Moritz AE, Foster JD, Gorentla BK et al (2013) Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding. J Biol Chem 288:20–32CrossRefPubMedGoogle Scholar
  48. 48.
    Buchmayer F, Schicker K, Steinkellner T et al (2013) Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate. Proc Natl Acad Sci U S A 110:11642–11647CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Hamilton PJ, Belovich AN, Khelashvili G et al (2014) PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein. Nat Chem Biol 10:582–589CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Scholze P, Freissmuth M, Sitte H (2002) Mutations within an intramembrane leucine heptad repeat disrupt oligomer formation of the rat GABA transporter 1. J Biol Chem 277:43682–43690CrossRefPubMedGoogle Scholar
  51. 51.
    Chiu CS, Jensen K, Sokolova I et al (2002) Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions. J Neurosci 22:10251–10266PubMedGoogle Scholar
  52. 52.
    Loland CJ, Norregaard L, Litman T et al (2002) Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proc Natl Acad Sci U S A 99:1683–1688CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Meinild A, Sitte H, Gether U (2004) Zinc potentiates an uncoupled anion conductance associated with the dopamine transporter. J Biol Chem 279:49671–49679CrossRefPubMedGoogle Scholar
  54. 54.
    Scholze P, Sitte H, Singer E (2001) Substantial loss of substrate by diffusion during uptake in HEK-293 cells expressing neurotransmitter transporters. Neurosci Lett 309:173–176CrossRefPubMedGoogle Scholar
  55. 55.
    Rosenauer R, Luf A, Holy M et al (2013) A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content. ACS Chem Neurosci 4:182–190CrossRefPubMedGoogle Scholar
  56. 56.
    Baumann MH, Partilla JS, Lehner KR et al (2013) Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive ‘bath salts’ products. Neuropsychopharmacology 38:552–562CrossRefPubMedGoogle Scholar
  57. 57.
    Sitte HH, Freissmuth M (2010) The reverse operation of Na(+)/Cl(−)-coupled neurotransmitter transporters—why amphetamines take two to tango. J Neurochem 112:340–355CrossRefPubMedGoogle Scholar
  58. 58.
    Mollenhauer HH, Morre DJ, Rowe LD (1990) Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochim Biophys Acta 1031:225–246CrossRefPubMedGoogle Scholar
  59. 59.
    Sitte HH, Scholze P, Schloss P et al (2000) Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study. J Neurochem 74:1317–1324CrossRefPubMedGoogle Scholar
  60. 60.
    Chattopadhyay A, Rukmini R, Mukherjee S (1996) Photophysics of a neurotransmitter: ionization and spectroscopic properties of serotonin. Biophys J 71:1952–1960CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Scholze P, Zwach J, Kattinger A et al (2000) Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter. J Pharmacol Exp Ther 293:870–878PubMedGoogle Scholar
  62. 62.
    Koepsell H, Lips K, Volk C (2007) Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res 24:1227–1251CrossRefPubMedGoogle Scholar
  63. 63.
    Courousse T, Gautron S (2015) Role of organic cation transporters (OCTs) in the brain. Pharmacol Ther 146:94–103CrossRefPubMedGoogle Scholar
  64. 64.
    Cui M, Aras R, Christian WV et al (2009) The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway. Proc Natl Acad Sci U S A 106:8043–8048CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Iversen LL (1997) The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: a novel catechol amine uptake process. 1964. Br J Pharmacol 120:267–282, discussion 264–266CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Vialou V, Balasse L, Callebert J et al (2008) Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice. J Neurochem 106:1471–1482PubMedGoogle Scholar
  67. 67.
    Kristufek D, Rudorfer W, Pifl C et al (2002) Organic cation transporter mRNA and function in the rat superior cervical ganglion. J Physiol 543:117–134CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Singer EA (1988) Transmitter release from brain slices elicited by single pulses: a powerful method to study presynaptic mechanisms. Trends Pharmacol Sci 9:274–276CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Thomas Steinkellner
    • 1
  • Felix P. Mayer
    • 1
  • Tina Hofmaier
    • 1
  • Marion Holy
    • 1
  • Therese Montgomery
    • 1
    • 2
  • Birgit Eisenrauch
    • 1
  • Michael Freissmuth
    • 1
  • Harald H. Sitte
    • 1
    Email author
  1. 1.Center of Physiology and Pharmacology, Institute of PharmacologyMedical University of ViennaViennaAustria
  2. 2.School of Biomolecular and Biomedical ScienceUniversity College DublinDublinIreland

Personalised recommendations