Galanthamine and Non-competitive Inhibitor Binding to ACh-binding Protein: Evidence for a Binding Site on Non-α-subunit Interfaces of Heteromeric Neuronal Nicotinic Receptors

Rapid neurotransmission is mediated through a superfamily of Cys-loop receptors that includes the nicotinic acetylcholine (nAChR), γ-aminobutyric acid (GABAA), serotonin (5-HT3) and glycine receptors. A class of ligands, including galanthamine, local anesthetics and certain toxins, interact with nAChRs non-competitively. Suggested modes of action include blockade of the ion channel, modulation from undefined extracellular sites, stabilization of desensitized states, and association with annular or boundary lipid. Alignment of mammalian Cys-loop receptors shows aromatic residues, found in the acetylcholine or ligand-binding pocket of nAChRs, are conserved in all subunit interfaces of neuronal nAChRs, including those that are not formed by α subunits on the principal side of the transmitter binding site. The amino-terminal domain containing the ligand recognition site is homologous to the soluble acetylcholine-binding protein (AChBP) from mollusks, an established structural and functional surrogate. We assess ligand specificity and employ X-ray crystallography with AChBP to demonstrate ligand interactions at subunit interfaces lacking vicinal cysteines (i.e. the non-α subunit interfaces in nAChRs). Non-competitive nicotinic ligands bind AChBP with high affinity (Kd 0.015–6 μM). We mutated the vicinal cysteine residues in loop C of AChBP to mimic the non-α subunit interfaces of neuronal nAChRs and other Cys loop receptors. Classical nicotinic agonists show a 10–40-fold reduction in binding affinity, whereas binding of ligands known to be non-competitive are not affected. X-ray structures of cocaine and galanthamine bound to AChBP (1.8 Å and 2.9 Å resolution, respectively) reveal interactions deep within the subunit interface and the absence of a contact surface with the tip of loop C. Hence, in addition to channel blocking, non-competitive interactions with heteromeric neuronal nAChR appear to occur at the non-α subunit interface, a site presumed to be similar to that of modulating benzodiazepines on GABAA receptors.