An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors.
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.
Grutter
Thomas
T
Institut Pasteur, URA 2182 CNRS 'Récepteurs et Cognition', Département des Biotechnologies, 25 rue du Dr Roux, 75724 Paris cedex 15, France, grutter@pasteur.fr or changeux@pasteur.fr
Prado de Carvalho
Lia
L
Le Novère
Nicolas
N
Corringer
Pierre Jean
PJ
Edelstein
Stuart
S
Changeux
Jean-Pierre
JP
D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002038:Bungarotoxins / Q000378:metabolism; D006860:Hydrogen Bonding; D007457:Iodine Radioisotopes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011725:Pyridines / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014316:Tritium
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; D007457:Iodine Radioisotopes; D011725:Pyridines; D011978:Receptors, Nicotinic; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D014316:Tritium; C082748:epibatidine; D000109:Acetylcholine