The Positive Allosteric Modulator Morantel Binds at Noncanonical Subunit Interfaces of Neuronal Nicotinic Acetylcholine Receptors

Abstract

We are interested in the positive allosteric modulation of neuronal nicotinic acetylcholine (ACh) receptors and have recently shown that the anthelmintic compound morantel potentiates by enhancing channel gating of the ␣3␤2 subtype. Based on the demonstration that morantelelicited currents were inhibited by the classic ACh competitor dihydro-␤-erythroidine in a noncompetitive manner and that morantel still potentiates at saturating concentrations of agonist (Wu et al., 2008), we hypothesized that morantel binds at the noncanonical ␤2(ϩ)/ ␣3(Ϫ) subunit interface. In the present study, we created seven cysteine-substituted subunits by site-directed mutagenesis, choosing residues in the putative morantel binding site with the aid of structural homology models. We coexpressed the mutant subunits and their respective wild-type partners in Xenopus oocytes and characterized the morantel potentiation of ACh-evoked currents, as well as morantel-evoked currents, before and after treatment with a variety of methanethiosulfonate (MTS)-based compounds, using voltageclamp recordings. The properties of four of the seven mutants, two residues on each side of the interface, were changed by MTS treatments. Coapplication with ACh enhanced the extent of MTS modification for ␣3A106C␤2 and ␣3␤2S192C receptors. The activities of two mutants, ␣3T115C␤2 and ␣3␤2T150C, were dramatically altered by MTS modification. For ␣3␤2T150C, while peak current amplitudes were reduced, potentiation was enhanced. For ␣3T115C␤2, both current amplitudes and potentiation were reduced. MTS modification and morantel were mutually inhibitory: MTS treatment decreased morantel-evoked currents and morantel decreased the rate of MTS modification. We conclude that the four residues showing MTS effects contribute to the morantel binding site.

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

“…Within the TMD, an intrasubunit PAM binding site in a7 nAChR was identified by mutational analyses (Young et al, 2008;daCosta et al, 2011), and photoaffinity labeling identified an intersubunit site in muscle-type nAChR (Nirthanan et al, 2008). Within the ECD, PAM binding sites were identified in secondary pockets at the agonist-binding canonical interfaces (Ludwig et al, 2010;Hamouda et al, 2013), at noncanonical interfaces (Seo et al, 2009;Hamouda et al, 2013;Olsen et al, 2014), and near the M2-M3 loop (Bertrand et al, 2008).…”

Structural and Functional Studies of the Modulator NS9283 Reveal Agonist-like Mechanism of Action at α4β2 Nicotinic Acetylcholine Receptors

“…On nAChRs, the described type of modulation may adhere to other modulators, including galanthamine and morantel, which all enhance potency through what is thought to be extracellular binding sites (16,33,41). Similarly, benzodiazepines acting on GABA A Rs also produce potentiation of GABAevoked currents without an increase in maximum efficacy.…”

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

“…This indicates a shared mechanism of action with other modulators of Cys-loop receptors that bind in non-canonical ECD interfaces. The most prominent example is benzodiazepine modulation at GABA A Rs (10), but interface binding sites have also been proposed for galanthamine and morantel (14,15). Whereas the case of NS9283 appears similar to that of benzodiazepines there is one important difference: the ␣␣-interface binds ACh and is hence canonical.…”

“…In GABA A Rs, the ␣␥-subunit extracellular domain (ECD) interface contains the binding site for benzodiazepines in a position homologous to the ␤␣-subunit agonist binding sites for GABA (10). Similar non-orthosteric binding sites in the nAChR ECD have also been proposed for galanthamine (14) and the ␣3␤4 nAChR modulator morantel (15). On the other hand etomidate has been shown to bind in the transmembrane domain (TMD) of GABA A Rs (16).…”