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Pharmacological and functional comparisons of α6/α3β2β3-nAChRs and α4β2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line

Acta Pharmacologica Sinicavolume 39pages15711581 (2018) | Download Citation



Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4β2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with β2 and β3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4β2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-β-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4β2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.

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  1. 1

    Jensen AA, Frolund B, Liljefors T, Krogsgaard-Larsen P. Neuronal nicotinic acetylcholine receptors: structural revelations, target identifications, and therapeutic inspirations. J Med Chem 2005; 48: 4705–45.

  2. 2

    Lukas RJ, Changeux JP, Le Novere N, Albuquerque EX, Balfour DJ, Berg DK, et al. International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 1999; 51: 397–401.

  3. 3

    Wu J, Lukas RJ. Naturally-expressed nicotinic acetylcholine receptor subtypes. Biochem Pharmacol 2011; 82: 800–7.

  4. 4

    Azam L, Winzer-Serhan UH, Chen Y, Leslie FM. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. J Comp Neurol 2002; 444: 260–74.

  5. 5

    Klink R, de Kerchove d'Exaerde A, Zoli M, Changeux JP. Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. J Neurosci 2001; 21: 1452–63.

  6. 6

    Shytle RD, Silver AA, Wilkinson BJ, Sanberg PR. A pilot controlled trial of transdermal nicotine in the treatment of attention deficit hyperactivity disorder. World J Biol Psychiatry 2002; 3: 150–5.

  7. 7

    Gerzanich V, Kuryatov A, Anand R, Lindstrom J. “Orphan” alpha6 nicotinic AChR subunit can form a functional heteromeric acetylcholine receptor. Mol Pharmacol 1997; 51: 320–7.

  8. 8

    Kuryatov A, Olale F, Cooper J, Choi C, Lindstrom J. Human alpha6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 2000; 39: 2570–90.

  9. 9

    Grinevich VP, Letchworth SR, Lindenberger KA, Menager J, Mary V, Sadieva KA, et al. Heterologous expression of human {alpha}6{beta}4{beta}3{alpha}5 nicotinic acetylcholine receptors: binding properties consistent with their natural expression require quaternary subunit assembly including the {alpha}5 subunit. J Pharmacol Exp Ther 2005; 312: 619–26.

  10. 10

    Dash B, Bhakta M, Chang Y, Lukas RJ. Modulation of recombinant, alpha2*, alpha3* or alpha4*-nicotinic acetylcholine receptor (nAChR) function by nAChR beta3 subunits. J Neurochem 2012; 121: 349–61.

  11. 11

    Tumkosit P, Kuryatov A, Luo J, Lindstrom J. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines. Mol Pharmacol 2006; 70: 1358–68.

  12. 12

    Boorman JP, Groot-Kormelink PJ, Sivilotti LG. Stoichiometry of human recombinant neuronal nicotinic receptors containing the b3 subunit expressed in Xenopus oocytes. J Physiol 2000; 529: 565–77.

  13. 13

    Broadbent S, Groot-Kormelink PJ, Krashia PA, Harkness PC, Millar NS, Beato M, et al. Incorporation of the beta3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors. Mol Pharmacol 2006; 70: 1350–7.

  14. 14

    Capelli AM, Castelletti L, Chen YH, Van der Keyl H, Pucci L, Oliosi B, et al. Stable expression and functional characterization of a human nicotinic acetylcholine receptor with alpha6beta2 properties: discovery of selective antagonists. Br J Pharmacol 2011; 163: 313–29.

  15. 15

    Dash B, Chang Y, Lukas RJ. Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) alpha5 Subunits and/or variants modulate function of alpha6*-nAChR. J Biol Chem 2011; 286: 37905–18.

  16. 16

    Dash B, Bhakta M, Chang Y, Lukas RJ. Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) alpha6 subunits that influence effects of wild-type or mutant beta3 subunits on function of alpha6beta2*- or alpha6beta4*-nAChR. J Biol Chem 2011; 286: 37976–89.

  17. 17

    Drenan RM, Grady SR, Whiteaker P, McClure-Begley T, McKinney S, Miwa JM, et al. In vivo activation of midbrain dopamine neurons via sensitized, high-affinity alpha 6 nicotinic acetylcholine receptors. Neuron 2008; 60: 123–36.

  18. 18

    Engle SE, Shih PY, McIntosh JM, Drenan RM. alpha4alpha6beta2* nicotinic acetylcholine receptor activation on ventral tegmental area dopamine neurons is sufficient to stimulate a depolarizing conductance and enhance surface AMPA receptor function. Mol Pharmacol 2013; 84: 393–406.

  19. 19

    Berry JN, Engle SE, McIntosh JM, Drenan RM. alpha6-Containing nicotinic acetylcholine receptors in midbrain dopamine neurons are poised to govern dopamine-mediated behaviors and synaptic plasticity. Neuroscience 2015; 304: 161–75.

  20. 20

    Henderson BJ, Wall TR, Henley BM, Kim CH, Nichols WA, Moaddel R, et al. Menthol alone upregulates midbrain nAChRs, alters nAChR subtype stoichiometry, alters dopamine neuron firing frequency, and prevents nicotine reward. J Neurosci 2016; 36: 2957–74.

  21. 21

    Drenan RM, Grady SR, Steele AD, McKinney S, Patzlaff NE, McIntosh JM, et al. Cholinergic modulation of locomotion and striatal dopamine release is mediated by alpha6alpha4* nicotinic acetylcholine receptors. J Neurosci 2010; 30: 9877–89.

  22. 22

    Yang K, Buhlman L, Khan GM, Nichols RA, Jin G, McIntosh JM, et al. Functional nicotinic acetylcholine receptors containing alpha6 subunits are on GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons. J Neurosci 2011; 31: 2537–48.

  23. 23

    Breining SR, Melvin M, Bhatti BS, Byrd GD, Kiser MN, Hepler CD, et al. Structure-activity studies of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes: a novel class of highly potent nicotinic receptor ligands. J Med Chem 2012; 55: 9929–45.

  24. 24

    Eaton JB, Peng JH, Schroeder KM, George AA, Fryer JD, Krishnan C, et al. Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells. Mol Pharmacol 2003; 64: 1283–94.

  25. 25

    Gentry CL, Lukas RJ. Local anesthetics noncompetitively inhibit function of four distinct nicotinic acetylcholine receptor subtypes. J Pharmacol Exp Ther 2001; 299: 1038–48.

  26. 26

    Wu J, Liu Q, Yu K, Hu J, Kuo YP, Segerberg M, et al. Roles of nicotinic acetylcholine receptor beta subunits in function of human alpha4-containing nicotinic receptors. J Physiol 2006; 576: 103–18.

  27. 27

    Wu J, Kuo YP, George AA, Xu L, Hu J, Lukas RJ. beta-Amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells. J Biol Chem 2004; 279: 37842–51.

  28. 28

    Zhao L, Kuo YP, George AA, Peng JH, Purandare MS, Schroeder KM, et al. Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharmacol Exp Ther 2003; 305: 1132–41.

  29. 29

    Huguenard JR, Prince DA. A novel T-type current underlies prolonged Ca2+-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 1992; 12: 3804–17.

  30. 30

    McIntosh JM, Azam L, Staheli S, Dowell C, Lindstrom JM, Kuryatov A, et al. Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors. Mol Pharmacol 2004; 65: 944–52.

  31. 31

    Yang KC, Jin GZ, Wu J. Mysterious alpha6-containing nAChRs: function, pharmacology, and pathophysiology. Acta Pharmacol Sin 2009; 30: 740–51.

  32. 32

    Vailati S, Hanke W, Bejan A, Barabino B, Longhi R, Balestra B, et al. Functional alpha6-containing nicotinic receptors are present in chick retina. Mol Pharmacol 1999; 56: 11–9.

  33. 33

    Dash B, Li MD, Lukas RJ. Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) beta3 subunits in enhanced functional expression of mouse alpha6beta2beta3- and alpha6beta4beta3-nAChRs. J Biol Chem 2014; 289: 28338–51.

  34. 34

    Rasmussen AH, Strobaek D, Dyhring T, Jensen ML, Peters D, Grunnet M, et al. Biophysical and pharmacological characterization of alpha6-containing nicotinic acetylcholine receptors expressed in HEK293 cells. Brain Res 2014; 1542: 1–11.

  35. 35

    Jensen AB, Hoestgaard-Jensen K, Jensen AA. Pharmacological characterisation of alpha6beta4 nicotinic acetylcholine receptors assembled from three chimeric alpha6/alpha3 subunits in tsA201 cells. Eur J Pharmacol 2014; 740: 703–13.

  36. 36

    Ley CK, Kuryatov A, Wang J, Lindstrom JM. Efficient expression of functional (alpha6beta2)2beta3 AChRs in Xenopus oocytes from free subunits using slightly modified alpha6 subunits. PLoS One 2014; 9: e103244.

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Work toward this project was supported by NIH R01 DA035958, NIH R21 DA026627, NIH R01 GM103801, P01 GM48677, R01 DA042749, Barrow Neuroscience Foundation, Philips Morris External Research Grant, and Special Innovation Project of Education Department of Guangdong Province. Production of the α6/3β2β3-nAChR cell line was sponsored by Targacept.

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Author notes

  1. These authors contributed equally to this work.


  1. Department of Neurology, Yunfu People’s Hospital, Yunfu, 527300, China

    • De-jie Chen
    • , Yuan-bing Huang
    • , Quang-xi Su
    •  & Jie Wu
  2. Department of Neurobiology, Barrow Neurological Institute, St Joseph’s Hospital and Medical Center, Phoenix, AZ, 85013, USA

    • De-jie Chen
    • , Fen-fei Gao
    • , Xiao-kuang Ma
    • , Yuan-bing Huang
    • , Ming Gao
    • , Turner Dharshaun
    • , Jason Brek Eaton
    • , Yong-chang Chang
    • , Ronald J Lukas
    • , Paul Whiteaker
    •  & Jie Wu
  3. Department of Pharmacology, Shantou University Medical College, Shantou, 515063, China

    • Fen-fei Gao
    • , Xiao-kuang Ma
    • , Gang-gang Shi
    •  & Jie Wu
  4. Departments of Psychology and Developmental Biology, Brigham Young University, Provo, UT, 84602, USA

    • Sterling Sudweeks
  5. George E Wahlen Veterans Affairs Medical Center, Salt Lake City, UT, 84108, USA

    • J Michael Mcintosh
  6. Departments of Psychiatry and Biology, University of Utah, Salt Lake City, UT, 84112, USA

    • J Michael Mcintosh
  7. Department of Physiology and Neuroscience, Brigham Young University, Provo, UT, 84602, USA

    • Scott C Steffensen


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Correspondence to Jie Wu.

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