Abstract
The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity “canonical” acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity “noncanonical” ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod–Wyman–Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.
Highlights
Neuronal nicotinic acetylcholine receptors are pentameric ligand-gated ion channels formed of identical or distinct but homologous subunits (α2–α10 and β2–β4)
We investigated the properties of CMPI and other nicotinic acetylcholine receptor (nAChR) ligands to delineate the pharmacology of the α4:α4 binding site and to elucidate allosteric interaction between the α4:β2 and α4:α4 interface-binding sites
To expand these studies to other nAChR agonists and to mechanistically characterize the interaction between drugs that bind at the α4:α4 agonist binding site (ABS) and agonists that interact with the α4:β2 sites, we first determined the effect of 1 μM CMPI on (α4)3(β2)2 nAChR currents induced by subsaturating or saturating concentrations of a series of nAChR agonists (Fig. 1)
Summary
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels formed of identical or distinct but homologous subunits (α2–α10 and β2–β4). The pharmacology of CMPI and NS9283 (location of binding site and effect on ACh concentration-response curve) at the (α4) 3(β2)2 nAChR resembles that of benzodiazepines at the GABAA receptor [27]. It binds to the α4:α4 interface with a higher affinity than ACh, cytisine, or nicotine, and efficaciously potentiates receptor responses to subsaturating concentrations of these agonists.
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