Tethered Agonist Analogs as Site-Specific Probes for Domains of the Human α7 Nicotinic Acetylcholine Receptor that Differentially Regulate Activation and Desensitization

Abstract

Homomeric ␣7 nicotinic acetylcholine receptors represent an important and complex pharmaceutical target. They can be activated by structurally diverse agonists and are highly likely to enter and remain in desensitized states at rates determined by the structures of the agonists. To identify structural elements regulating this function, we introduced reactive cysteines into the ␣7 ligand-binding domain allowing us to bind sulfhydrylreactive (SH) agonist analogs or control reagents onto specific positions in the ligand binding domain. We identified four ␣7 mutants (S36C, L38C, W55C, and L119C) in which the tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor. However, after selective reaction with SH agonist analogs, the type II allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-NЈ-(5-methyl-3-isoxazolyl-3-isoxazolyl)-urea (PNU-120596) could reactivate L119C and W55C mutants and receptors with a reduced or modified C-loop. Modified S36C and L38C mutants were insensitive to reactivation by PNU-120596, whether they were reacted with agonist analogs or alternative SH reagents. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in a similar but nonidentical position underneath the C-loop. The orientation assumed by the ligand tethered to 119C was approximately 3-fold more sensitive to PNU-120596 than the alternative pose at 55C. Our results support the hypothesis that a single ligand can bind within the receptor in different ways and, depending on the specific binding pose, may variously promote activation or desensitization, or, alternatively, function as a competitive antagonist. This insight may provide a new approach for drug development.

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

“…Although MLA is considered to be a competitive antagonist of orthosteric agonists, as reported previously (20), we found that the inhibition of GAT107 direct activation was not surmountable by increasing concentrations of GAT107 (data not shown). This is consistent with MLA acting as a sort of an inverse agonist, as we have previously seen when it was applied to receptors with tethered agonists (29). Although this effect of MLA is consistent with GAT107 not producing direct activation by binding to the orthosteric site, it is not sufficient to prove that GAT107 is working exclusively at a single allosteric site to produce both potentiation and direct activation.…”

Investigation of the Molecular Mechanism of the α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator PNU-120596 Provides Evidence for Two Distinct Desensitized States

“…The structure of the acetylcholine binding protein bound with 3-(2,4-dimethoxybenzylidene)anabaseine, the compound that most effectively induced D i or other nonresponsive states, showed that the molecule might be bound in entirely different orientations at the various subunit interfaces (Hibbs et al, 2009). In addition, recent work with tethered agonists has shown that a single ligand molecule may bind the ligand binding domain in different orientations, each of which may differentially affect activation and desensitization (Wang et al, 2010). Identification of compounds that preferentially stabilize specific functional states, including D s and D i , may be therapeutically important if it can be shown that a specific state is important for signal transduction attributed to ␣7.…”

Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

“…Covalent trapping of a reactive analog at an introduced cysteine has previously been used to integrate computational techniques such as homology modeling and molecular dynamics, with functional analysis of the trapped ligand-receptor complex (29,40). Here we have used this method for a different purpose, to reveal the interfacial binding site of a ligand in a heteropentameric nAChR.…”