Classical Radioligand Uptake and Binding Methods in Transporter Research: An Emphasis on the Monoamine Neurotransmitter Transporters

  • Sonja SucicEmail author
  • Heinz Bönisch
Part of the Neuromethods book series (NM, volume 118)


Radioligand uptake and binding assays denote an invaluable tool in the field of neurotransmitter transporter research. Their benefits have been evident since the late 1950s, and they continue to contribute major insights into transporter function and structure to date. In the current chapter, we focus primarily on the family of monoamine (MA) neurotransmitter transporters (MATs), i.e., transporters (T) for norepinephrine [noradrenaline] (NET), dopamine (DAT), and serotonin [5-hydroxy tryptamine, 5-HT] (SERT), dysfunction of which has been linked to numerous neuropsychiatric disorders and substance abuse. Radiotracer assays have provided a major way of elucidating the mechanisms of action of not only endogenous substrates (e.g., NE, SER, and DA), but also many diverse substances such as antidepressants (e.g., imipramine and citalopram), psychostimulants (e.g., amphetamine and cocaine), toxins (e.g., conotoxins), or neurotoxins (e.g., 1-methyl-4-phenylpyridinium, MPP+) that exert their action on MATs. In this chapter we describe the basic principles and experimental procedures of radiotracer assays commonly used in the studies of MATs.

Key words

Radioligands Uptake Binding Monoamine neurotransmitter transporters 


  1. 1.
    Von Euler US (1946) The presence of a sympathomimetic substance in extracts of mammalian heart. J Physiol 105:38–44CrossRefGoogle Scholar
  2. 2.
    Axelrod J, Weil-Malherbe H, Tomchick R (1959) The physiological disposition of H3-epinephrine and its metabolite metanephrine. J Pharmacol Exp Ther 127:251–256PubMedGoogle Scholar
  3. 3.
    Whitby LG, Hertting G, Axelrod J (1960) Effect of cocaine on the disposition of noradrenaline labelled with tritium. Nature 187:604–605CrossRefPubMedGoogle Scholar
  4. 4.
    Herting G, Axelrod J, Whitby LG (1961) Effect of drugs on the uptake and metabolism of H3-norepinephrine. J Pharmacol Exp Ther 134:146–153PubMedGoogle Scholar
  5. 5.
    Dengler HJ, Spiegel HE, Titus EO (1961) Uptake of tritium-labeled norepinephrine in brain and other tissues of cat in vitro. Science 133(3458):1072–1073CrossRefPubMedGoogle Scholar
  6. 6.
    Dengler HJ, Michaelson IA, Spiegel HE, Titus E (1962) The uptake of labelled norepinephrine by isolated brain and other tissues of the cat. Int J Neuropharmacol 1:23–38CrossRefGoogle Scholar
  7. 7.
    Iversen LL (1963) The uptake of norepinephrine by the isolated perfused rat heart. Br J Pharmacol 21:523–537Google Scholar
  8. 8.
    Iversen LL (1967) The uptake and storage of noradrenaline in sympathetic nerves. Cambridge University Press, Cambridge, UKGoogle Scholar
  9. 9.
    Langeloh A, Bönisch H, Trendelenburg U (1987) The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: multifactorial induction of outward transport. Naunyn Schmiedebergs Arch Pharmacol 336:602–610CrossRefPubMedGoogle Scholar
  10. 10.
    Bönisch H (1984) The transport of (+)-amphetamine by the neuronal noradrenaline carrier. Naunyn Schmiedebergs Arch Pharmacol 327:267–272CrossRefPubMedGoogle Scholar
  11. 11.
    Bönisch H, Brüss M (2006) The norepinephrine transporter in physiology and disease. Handb Exp Pharmacol 175:485–524CrossRefPubMedGoogle Scholar
  12. 12.
    Pacholczyk T, Blakely RD, Amara SG (1991) Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 350(6316):350–354CrossRefPubMedGoogle Scholar
  13. 13.
    Raisman R, Briley MS, Langer SZ (1980) Specific tricyclic antidepressant binding sites in rat brain characterised by high-affinity 3H-imipramine binding. Eur J Pharmacol 61:373–380CrossRefPubMedGoogle Scholar
  14. 14.
    Pramod AB, Foster Carvelli JL, Henry LK (2013) SLC6 transporters: structure, function, regulation, disease association and therapeutics. Mol Aspects Med 34:197–219CrossRefPubMedGoogle Scholar
  15. 15.
    Penmatsa A, Wang KH, Gouaux E (2013) X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature 503(7474):85–90CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wang KH, Penmatsa A, Gouaux E (2015) Neurotransmitter and psychostimulant recognition by the dopamine transporter. Nature 521(7552):322–327CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Rudnick G, Krämer R, Blakely RD, Murphy DL, Verrey F (2014) The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction. Pflugers Arch 466:25–42CrossRefPubMedGoogle Scholar
  18. 18.
    Trendelenburg U (1990) Carrier-mediated outward transport of noradrenaline from adrenergic varicosities. Pol J Pharmacol Pharm 42:515–520CrossRefPubMedGoogle Scholar
  19. 19.
    Cheng Y, Prusoff W (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108CrossRefPubMedGoogle Scholar
  20. 20.
    Bönisch H, Harder R (1986) Binding of 3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells). Naunyn Schmiedebergs Arch Pharmacol 334:403–411CrossRefPubMedGoogle Scholar
  21. 21.
    Schömig E, Bönisch H (1986) Solubilization and characterization of the 3H-desipramine binding site of rat phaeochromocytoma cells (PC12-cells). Naunyn Schmiedebergs Arch Pharmacol 334:412–417CrossRefPubMedGoogle Scholar
  22. 22.
    Bönisch H (1998) Transport and drug binding kinetics in membrane vesicle preparations. In: Amara S (ed). Neurotransmitter transporters. Methods Enzymol 296:259–278Google Scholar
  23. 23.
    Eshleman AJ, Stewart E, Evenson AK, Mason JN, Blakely RD, Janowsky A, Neve KA (1997) Metabolism of catecholamines by catechol-O-methyltransferase in cells expressing recombinant catecholamine transporters. J Neurochem 69:1459–1466CrossRefPubMedGoogle Scholar
  24. 24.
    Bönisch H, Rodrigues-Pereira E (1983) Uptake of 14C-tyramine and release of extravesicular 3H-noradrenaline in isolated perfused rabbit hearts. Naunyn Schmiedebergs Arch Pharmacol 323:233–244CrossRefPubMedGoogle Scholar
  25. 25.
    Wenge B, Bönisch H (2013) The role of cysteines and histidins of the norepinephrine transporter. Neurochem Res 38:1303–1314CrossRefPubMedGoogle Scholar
  26. 26.
    Yamamura HI, Enna SJ, Kuhar MJ (1978) Neurotransmitter receptor binding. Raven Press, New YorkGoogle Scholar
  27. 27.
    Williams LT, Lefkowitz RJ (1978) Receptor binding studies in adrenergic pharmacology. Raven Press, New YorkGoogle Scholar
  28. 28.
    Reith MEA (1997) Neurotransmitter transporters (ed.). Structure, function, and regulation. Humana, Totowa, NJGoogle Scholar
  29. 29.
    Amara SG (1998) Neurotransmitter transporters (ed). Methods Enzymol 296:307-318Google Scholar
  30. 30.
    Janowsky A, Neve K, Eshleman AJ (2001) Uptake and release of neurotransmitters. Curr Protoc Neurosci Chapter 7:Unit 7.9Google Scholar
  31. 31.
    Motulsky HJ, Christopoulos A (2003) Fitting models to biological data using linear and nonlinear regression. A practical guide to curve fitting. GraphPad Sostware Inc., San Diego, CA,
  32. 32.
    Sitte HH, Freissmuth M (2006) Neurotransmitter transporters (eds). Handb Exp Pharmacol, vol 175. Springer, BerlinGoogle Scholar
  33. 33.
    Iversen LL (1965) The inhibition of noradrenaline uptake by drugs. Adv Drug Res 2:1–46PubMedGoogle Scholar
  34. 34.
    Iversen LL (1965) The uptake of adrenaline by the rat isolated heart. Br J Pharmacol 24:387–394Google Scholar
  35. 35.
    Burgen ASV, Iversen LL (1965) The inhibition of noradrenaline uptake by sympathomimetic amines in the rat isolated heart. Br J Pharmacol 25:34–49Google Scholar
  36. 36.
    Born GV, Gillson RE (1959) Studies on the uptake of 5-hydroxytryptamine by blood platelets. J Physiol 146(3):472–491CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Pifl C, Giros B, Caron MG (1993) Dopamine transporter expression confers cytotoxicity to low doses of the Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium. J Neurosci 13:4246–4253PubMedGoogle Scholar
  38. 38.
    Callingham BA (1967) The effects of imipramine and related compounds on the uptake of noradrenaline into sympathetic nerve endings. In: Garattini S, Dukes MNG (eds) Excerpta medica international congress series 122. Excerpta Medica Foundation, Amsterdam, pp 35–43Google Scholar
  39. 39.
    Maxwell RA, Ferris RM, Burcsu J, Chaplin Woodward E, Tang D, Williard K (1974) The phenyl rings of tricyclic antidepressants and related compounds as determinants of the potency of inhibition of the amine pumps in adrenergic neurons of the rabbit aorta and in rat cortical synaptosomes. J Pharmacol Exp Ther 191:418–430PubMedGoogle Scholar
  40. 40.
    Koe BK (1976) Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J Pharmacol Exp Ther 199:649–661PubMedGoogle Scholar
  41. 41.
    Richelson E, Pfenning M (1984) Blockade by antidepressants and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake. Eur J Pharmacol 104:277–286CrossRefPubMedGoogle Scholar
  42. 42.
    Segel IH (1975) Enzyme kinetics. Behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, New YorkGoogle Scholar
  43. 43.
    Harder R, Bönisch H (1985) Effects of monovalent ions on the transport of noradrenaline across the plasma membrane of neuronal cells (PC-12 cells). J Neurochem 45:1154–1162CrossRefPubMedGoogle Scholar
  44. 44.
    Schömig E, Korber M, Bönisch H (1988) Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier. Naunyn Schmiedebergs Arch Pharmacol 337:626–632PubMedGoogle Scholar
  45. 45.
    Ramamoorthy S, Ramamoorthy JD, Prasad PD, Bhat GK, Mahesh VB, Leibach FH, Ganapathy V (1995) Regulation of the human serotonin transporter by interleukin-1 beta. Biochem Biophys Res Commun 216(2):560–567CrossRefPubMedGoogle Scholar
  46. 46.
    Kong E, Sucic S, Monje FJ, Savalli G, Diao W, Khan D, Ronovsky M, Cabatic M, Koban F, Freissmuth M, Pollak DD (2015) STAT3 controls IL6-dependent regulation of serotonin transporter function and depression-like behavior. Sci Rep 5:9009CrossRefPubMedGoogle Scholar
  47. 47.
    Cool DR, Leibach FH, Bhalla VK, Mahesh VB, Ganapathy V (1991) Expression and cyclic AMP-dependent regulation of a high affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR). J Biol Chem 266(24):15750–15757PubMedGoogle Scholar
  48. 48.
    Qian Y, Galli A, Ramamoorthy S, Risso S, DeFelice LJ, Blakely RD (1997) Protein kinase C activation regulates human serotonin transporters in HEK293 cells via altered cell surface expression. J Neurosci 17(1):45–57PubMedGoogle Scholar
  49. 49.
    Apparsundaram S, Galli A, DeFelice LJ, Hartzell HC, Blakely RD (1998) Acute regulation of norepinephrine transport: I. Protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells. J Pharmacol Exp Ther 287:733–743PubMedGoogle Scholar
  50. 50.
    Sucic S, Bryan-Lluka LJ (2002) The role of the conserved GXXXRXG motif in the expression and function of the human norepinephrine transporter. Brain Res Mol Brain Res 108:40–50CrossRefPubMedGoogle Scholar
  51. 51.
    Sucic S, Packowski FA, Runkel F, Bönisch H, Bryan-Lluka LJ (2002) Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter. J Neurochem 81:344–354CrossRefPubMedGoogle Scholar
  52. 52.
    Paczkowski FA, Bryan-Lluka LJ (2001) Tyrosine residue 271 of the norepinephrine transporter is an important determinant of its pharmacology. Brain Res Mol Brain Res 97:32–42CrossRefPubMedGoogle Scholar
  53. 53.
    Paczkowski FA, Bönisch H, Bryan-Lluka LJ (2002) Pharmacological properties of the naturally occurring Ala457Pro variant of the human norepinephrine transporter. Pharmacogenetics 12:165–173CrossRefPubMedGoogle Scholar
  54. 54.
    Giros B, Wang Y-M, Suter S, McLeskey SB, Pifl C, Caron MG (1994) Delineation of discrete domains for substrate, cocaine, and tricyclic antidepressant interactions using chimeric dopamine-norepinephrine transporters. J Biol Chem 269:15985–15988PubMedGoogle Scholar
  55. 55.
    Buck KJ, Amara SG (1994) Chimeric dopamine-norepinephrine transporters delineate structural domains influencing selectivity for catecholamines and 1-methyl-4-phenylpyridinium. Proc Natl Acad Sci U S A 91:12584–12588CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Buck KJ, Amara SG (1995) Structural domains of catecholamine transporter chimeras involved in selective inhibition by antidepressants and psychomotor stimulants. Mol Pharmacol 48:1030–1037PubMedGoogle Scholar
  57. 57.
    Kitayama S, Shimada S, Xu H, Markham L, Donovan DM, Uhl GR (1992) Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc Natl Acad Sci U S A 89:7782–7785CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Barker EL, Blakely RD (1996) Identification of a single amino acid, phenylalanine 586, that is responsible for high affinity interactions of tricyclic antidepressants with the human serotonin transporter. Mol Pharmacol 50:957–965PubMedGoogle Scholar
  59. 59.
    Surratt CK, Ukairo OT, Ramanujapuram S (2005) Recognition of psychostimulants, antidepressants, and other inhibitors of synaptic neurotransmitter uptake by the plasma membrane monoamine transporters. AAPS J 7:E739–E751CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Stöber G, Nöthen MM, Pörzgen P, Brüss M, Bönisch H, Knapp M, Beckmann H, Propping P (1996) Systematic search for variation in the human norepinephrine transporter gene: identification of five naturally occurring missense mutations and study of association with major psychiatric disorders. Am J Med Genet 67:523–532CrossRefPubMedGoogle Scholar
  61. 61.
    Stöber G, Hebebrand J, Cichon S, Brüss M, Bönisch H, Lehmkuhl G, Poustka F, Schmidt M, Remschmidt H, Propping P, Nöthen MM (1999) Tourette syndrome and the norepinephrine transporter gene: results of a systematic mutation screening. Am J Med Genet 88:158–163CrossRefPubMedGoogle Scholar
  62. 62.
    Shannon JR, Flattem NL, Jordan J, Jacob G, Black BK, Biaggioni I, Blakely RD, Robertson D (2000) Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N Engl J Med 342:541–549CrossRefPubMedGoogle Scholar
  63. 63.
    Rodríguez GJ, Roman DL, White KJ, Nichols DE, Barker EL (2003) Distinct recognition of substrates by the human and Drosophila serotonin transporters. J Pharmacol Exp Ther 306(1):338–346CrossRefPubMedGoogle Scholar
  64. 64.
    Han DD, Gu HH (2006) Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs. BMC Pharmacol 6:6CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Sarker S, Weissensteiner R, Steiner I, Sitte HH, Ecker GF, Freissmuth M, Sucic S (2010) The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter. Mol Pharmacol 78:1026–1035CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Sucic S, Dallinger S, Zdrazil B, Weissensteiner R, Jorgensen TN, Holy M, Kudlacek O, Seidel S, Cha JH, Gether U, Newman AH, Ecker GF, Freissmuth M, Sitte HH (2010) The amino terminus of monoamine transporters is a lever required for the action of amphetamines. J Biol Chem 285:10924–10938CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Kitayama S, Mitsuhata C, Davis S, Wang J-B, Sato T, Morita K, Uhl GR, Dohi T (1998) MPP+ toxicity and plasma membrane dopamine transporter: study using cell lines expressing the wild-type and mutant rat dopamine transporters. Biochim Biophys Acta 1404:305–313CrossRefPubMedGoogle Scholar
  68. 68.
    Guptaroy B, Fraser R, Desai A, Zhang M, Gnegy ME (2001) Site-directed mutations near transmembrane domain 1 alter conformation and function of norepinephrine and dopamine transporters. Mol Pharmacol 79(3):520–532CrossRefGoogle Scholar
  69. 69.
    Tatsumi M, Groshan K, Blakely RD, Richelson E (1997) Pharmacological profile of antidepressants and related compounds at human monoamine transporters. Eur J Pharmacol 340:249–258CrossRefPubMedGoogle Scholar
  70. 70.
    Lynagh T, Khamu TS, Bryan-Lluka LJ (2014) Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity. Naunyn Schmiedebergs Arch Pharmacol 387:95–107CrossRefPubMedGoogle Scholar
  71. 71.
    Scholze P, Nørregaard L, Singer EA, Freissmuth M, Gether U, Sitte HH (2002) The role of zinc ions in reverse transport mediated by monoamine transporters. J Biol Chem 277(24):21505–21513CrossRefPubMedGoogle Scholar
  72. 72.
    Karlin A, Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293:123–145CrossRefPubMedGoogle Scholar
  73. 73.
    Javitch JA (1998) Probing structure of neurotransmitter transporters by substituted cysteine accessibility methods. Methods Enzymol 296:331–346CrossRefPubMedGoogle Scholar
  74. 74.
    Ferrer JV, Javitch JA (1998) Cocaine alters the accessibility of endogenous cysteines in putative extracellular and intracellular loops of the human dopamine transporter. Proc Natl Acad Sci U S A 95:9238–9243CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Chen J-G, Rudnick G (2000) Permeation and gating residues in serotonin transporter. Proc Natl Acad Sci U S A 97:1044–1049CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Sucic S, Bryan-Lluka LJ (2005) Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis. J Neurochem 94(6):1620–1630CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute of Pharmacology, Center of Physiology and PharmacologyMedical University of ViennaViennaAustria
  2. 2.Biomedical Center, Institute of Pharmacology and ToxicologyUniversity of BonnBonnGermany

Personalised recommendations