RESUMO
The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site but does not directly contact the ligand plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently, the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function) or if a compensating backbone mutation is introduced to relieve unfavorable electrostatics.
Assuntos
Ácido Aspártico/química , Sítios de Ligação , Receptores Nicotínicos/química , Sequência de Aminoácidos , Aminoácidos/química , Aminobutiratos/química , Animais , Simulação por Computador , Eletrofisiologia , Ligação de Hidrogênio , Ligantes , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Estrutura Terciária de Proteína , Receptores Nicotínicos/genética , Homologia Estrutural de Proteína , Triptofano/análogos & derivados , Triptofano/química , Xenopus laevisRESUMO
The binding of three distinct agonists-acetylcholine (ACh), nicotine, and epibatidine-to the nicotinic acetylcholine receptor has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp alpha149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond. A simple structural model that considers only possible interactions with Trp alpha149 suggests that a novel aromatic C-H...O=C hydrogen bond further augments the binding of epibatidine. These studies illustrate the subtleties and complexities of the interactions between drugs and membrane receptors and establish a paradigm for obtaining detailed structural information.
