An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

EMBO J. 2003 May 1;22(9):1990-2003. doi: 10.1093/emboj/cdg197.


The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced alpha4 residues into the alpha7/5HT(3) chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch-clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-affinity alpha-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric-kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in alpha4 and in alpha7 double mutant but not in alpha7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / metabolism*
  • Amino Acid Sequence
  • Binding Sites
  • Bridged Bicyclo Compounds, Heterocyclic / metabolism
  • Bungarotoxins / metabolism
  • Hydrogen Bonding
  • Iodine Radioisotopes
  • Models, Molecular
  • Molecular Sequence Data
  • Pyridines / metabolism
  • Receptors, Nicotinic / chemistry
  • Receptors, Nicotinic / metabolism*
  • Receptors, Serotonin / metabolism
  • Receptors, Serotonin, 5-HT3
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Tritium


  • Bridged Bicyclo Compounds, Heterocyclic
  • Bungarotoxins
  • Iodine Radioisotopes
  • Pyridines
  • Receptors, Nicotinic
  • Receptors, Serotonin
  • Receptors, Serotonin, 5-HT3
  • Recombinant Fusion Proteins
  • Tritium
  • epibatidine
  • Acetylcholine

Associated data

  • PDB/1I9B