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

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Oxford University Press

RESUMO

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 α4 residues into the α7/5HT3 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 α-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 α4 and in α7 double mutant but not in α7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

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