Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors


Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC50. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC50’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Benwell ME, Balfour DJ, Anderson JM (1988) Evidence that tobacco smoking increases the density of (−)-[3H]nicotine binding sites in human brain. J Neurochem 50:1243–1247. doi:10.1111/j.1471-4159.1988.tb10600.x

    PubMed  Article  CAS  Google Scholar 

  2. Breese CR, Marks MJ, Logel J, Adams CE, Sullivan B, Collins AC et al (1997) Effect of smoking history on [3H]nicotine binding in human postmortem brain. J Pharmacol Exp Ther 282:7–13

    PubMed  CAS  Google Scholar 

  3. Downing JE, Role L (1987) Activators of protein kinase C enhance acetylcholine receptor desensitization in sympathetic ganglion neurons. Proc Natl Acad Sci USA 84:7739–7743. doi:10.1073/pnas.84.21.7739

    PubMed  Article  CAS  Google Scholar 

  4. Eilers H, Schaeffer E, Bickler PE, Forsayeth JR (1997) Functional deactivation of the major neuronal nicotinic receptor caused by nicotine and a protein kinase C-dependent mechanism. Mol Pharmacol 52:1105–1112

    PubMed  CAS  Google Scholar 

  5. Fenster CP, Beckman ML, Parker JC, Sheffield EB, Whitworth TL, Quick MW et al (1999a) Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C. Mol Pharmacol 55:432–443

    PubMed  CAS  Google Scholar 

  6. Fenster CP, Whitworth TL, Sheffield EB, Quick MW, Lester RA (1999b) Upregulation of surface alpha4beta2 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine. J Neurosci 19:4804–4814

    PubMed  CAS  Google Scholar 

  7. Flores CM, Rogers SW, Pabreza LA, Wolfe BB, Kellar KJ (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits and is up-regulated by chronic nicotine treatment. Mol Pharmacol 41:31–37

    PubMed  CAS  Google Scholar 

  8. Huganir RL, Delcour AH, Greengard P, Hess GP (1986) Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature 321:774–776. doi:10.1038/321774a0

    PubMed  Article  CAS  Google Scholar 

  9. Ke L, Eisenhour CM, Bencherif M, Lukas RJ (1998) Effects of chronic nicotine treatment on expression of diverse nicotinic acetylcholine receptor subtypesI. Dose- and time-dependent effects of nicotine treatment. J Pharmacol Exp Ther 286:825–840

    PubMed  CAS  Google Scholar 

  10. Khiroug L, Sokolova E, Giniatullin R, Afzalov R, Nistri A (1998) Recovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers. J Neurosci 18:2458–2466

    PubMed  CAS  Google Scholar 

  11. Lapchack PA, Araujo DM, Quirion R, Collier B (1989) Effect of chronic nicotine treatment on nicotinic autoreceptor function and N-3H-Mcc binding sites in the rat brain. J Neurochem 52:483–491. doi:10.1111/j.1471-4159.1989.tb09146.x

    Article  Google Scholar 

  12. López-Hernández GY, Sánchez-Padilla J, Ortiz-Acevedo A, Lizardi-Ortiz J, Salas-Vincenty J, Rojas V et al (2004) Nicotine-induced up-regulation and desensitization of α4β2 neuronal nicotinic receptors depend on subunit ratio. J Biol Chem 279:38007–38015. doi:10.1074/jbc.M403537200

    PubMed  Article  Google Scholar 

  13. Lukas RJ (1991) Effects of chronic nicotinic ligand exposure on functional activity of nicotinic acetylcholine receptors expressed by cells of the PC12 rat pheochromocytoma or the TE671/RD human clonal line. J Neurochem 56:1134–1145. doi:10.1111/j.1471-4159.1991.tb11403.x

    PubMed  Article  CAS  Google Scholar 

  14. Marks MJ, Burch JB, Collins AC (1983) Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J Pharmacol Exp Ther 226:817–825

    PubMed  CAS  Google Scholar 

  15. Moss SJ, McDonald BJ, Rudhard Y, Schoepfer R (1996) Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor alpha 7 subunit by cAMP-dependent protein kinase. Neuropharmacology 35:1023–1028. doi:10.1016/S0028-3908(96)00083-4

    PubMed  Article  CAS  Google Scholar 

  16. Nakayama H, Okuda H, Nakashima T (1993) Phosphorylation of rat brain nicotinic acetylcholine receptor by cAMP-dependent protein kinase in vitro. Brain Res Mol Brain Res 20:171–177. doi:10.1016/0169-328X(93)90123-7

    PubMed  Article  CAS  Google Scholar 

  17. Schwartz RD, Kellar KJ (1985) In vivo regulation of [3H]acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J Neurochem 45:427–433. doi:10.1111/j.1471-4159.1985.tb04005.x

    PubMed  Article  CAS  Google Scholar 

  18. Swope SL, Moss SJ, Blackstone CD, Huganir RL (1992) Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB 6:2514–2523

    CAS  Google Scholar 

  19. Vijayaraghavan S, Schmid HA, Halvorsen SW, Berg DK (1990) Cyclic AMP-dependent phosphorylation of a neuronal acetylcholine receptor alpha-type subunit. J Neurosci 10:3255–3262

    PubMed  CAS  Google Scholar 

  20. Viseshakul N, Figl A, Lytle C, Cohen BN (1998) The alpha4 subunit of rat alpha4beta2 nicotinic receptors is phosphorylated in vivo. Brain Res Mol Brain Res 59:100–104. doi:10.1016/S0169-328X(98)00128-4

    PubMed  Article  CAS  Google Scholar 

  21. Wecker L, Guo X, Rycerz AM, Edwards SC (2001) Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits. J Neurochem 76:711–720. doi:10.1046/j.1471-4159.2001.00041.x

    PubMed  Article  CAS  Google Scholar 

  22. Whiteaker P, Sharples CG, Wonnacott S (1998) Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition. Mol Pharmacol 53:950–962

    PubMed  CAS  Google Scholar 

Download references


We thank Mr. José Serrano, Dr. Janice Salas, and Dr. Amelia Rivera for valuable technical assistance. This research was supported by National Institutes of Health Grants NIH 2RO1GM56371-11, SNRP-U54N54301, and GM08102-27. The Research Initiative for Scientific Enhancement-Minority Biomedical Research Support (RISE-MBRS)-NIH program (5R25GM61151) supported Gretchen Y. López-Hernández. Nilza M. Biaggi-Labiosa was supported by the RISE-MBRS-NIH program (2R25GM061151). Alexis Torres-Cintrón was supported by the Minority Access to Research Careers (MARC)-MBRS-NIH program.

Author information



Corresponding author

Correspondence to José A. Lasalde-Dominicci.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

López-Hernández, G.Y., Biaggi-Labiosa, N.M., Torres-Cintrón, A. et al. Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors. Cell Mol Neurobiol 29, 41–53 (2009).

Download citation


  • Whole-mount immunofluorescence assay
  • Epibatidine binding
  • Nicotine
  • Voltage-clamp
  • Confocal imaging