Engineering α4β2 nAChRs with reduced or increased nicotine sensitivity via selective disruption of consensus sites in the M3-M4 cytoplasmic loop of the α4 subunit

Neuropharmacology. 2015 Dec:99:273-84. doi: 10.1016/j.neuropharm.2015.04.022. Epub 2015 May 6.


The α4β2 neuronal nicotinic acetylcholine receptor (nAChR) plays a crucial role in nicotine addiction. These receptors are known to desensitize and up-regulate after chronic nicotine exposure, but the mechanism remains unknown. Currently, the structure and functional role of the intracellular domains of the nAChR are obscure. To study the effect of subunit phosphorylation on α4β2 nAChR function and expression, eleven residues located in the M3-M4 cytoplasmic loop were mutated to alanine and aspartic acid. Two-electrode voltage clamp and 125I-labeled epibatidine binding assays were performed on Xenopus oocytes to assess agonist activation and receptor expression. When ACh was used as an agonist, a decrease in receptor activation was observed for the majority of the mutations. When nicotine was used as an agonist, four mutations exhibited a statistically significant hypersensitivity to nicotine (S438D, S469A, Y576A, and S589A). Additionally, two mutations (S516D and T536A) that displayed normal activation with ACh displayed remarkable reductions in sensitivity to nicotine. Binding assays revealed a constitutive up-regulation in these two nicotine mutations with reduced nicotine sensitivity. These results suggest that consensus phosphorylation residues in the M3-M4 cytoplasmic loop of the α4 subunit play a crucial role in regulating α4β2 nAChR agonist selectivity and functional expression. Furthermore, these results suggest that disruption of specific interactions at PKC putative consensus sites can render α4β2 nAChRs almost insensitive to nicotine without substantial effects on normal AChR function. Therefore, these PKC consensus sites in the M3-M4 cytoplasmic loop of the α4 nAChR subunit could be a target for smoking cessation drugs.

Keywords: Nicotine; Nicotine sensitivity; Nicotinic acetylcholine receptor; Phosphorylation; Two-electrode voltage clamp.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / pharmacology
  • Animals
  • Bridged Bicyclo Compounds, Heterocyclic / pharmacology
  • Casein Kinase II / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cytoplasm
  • Iodine Radioisotopes
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Mutagenesis, Site-Directed
  • Mutation
  • Nicotinic Agonists / pharmacology*
  • Oocytes
  • Patch-Clamp Techniques
  • Phosphorylation
  • Protein Kinase C / metabolism
  • Pyridines / pharmacology
  • Rats
  • Receptors, Nicotinic / genetics*
  • Receptors, Nicotinic / metabolism*
  • Xenopus


  • Bridged Bicyclo Compounds, Heterocyclic
  • Iodine Radioisotopes
  • Nicotinic Agonists
  • Pyridines
  • Receptors, Nicotinic
  • nicotinic receptor alpha4beta2
  • Casein Kinase II
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • epibatidine
  • Acetylcholine