Allosteric Agonism of α7 Nicotinic Acetylcholine Receptors: Receptor Modulation Outside the Orthosteric Site

Abstract

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. Typically, channel activation follows the binding of agonists to the orthosteric binding sites of the receptor. a7 nAChRs have a very low probability of channel activation, which can be reversed by the binding of a7 selective positive allosteric modulators (PAMs) to putative sites within the transmembrane domains. Although typical PAMs, like PNU-120596, require coapplication of an orthosteric agonist to produce large channel activations, some, like GAT107 and B-973B [(S)-3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide], are characterized as allosteric activating PAMs, which also bind to an allosteric activation (AA) site in the extracellular domain and activate the a7 ion channel by themselves. We had previously characterized N,N-diethyl-N9-phenylpiperazine analogs with various functions. In this work, we docked members of this family to a homology model of the a7 receptor extracellular domain. The compound 1,1-diethyl-4(naphthalene-2-yl)piperazin-1-ium (2NDEP) a weak partial agonist, showed particularly favorable docking and binding energies at the putative AA site of the receptor. We hypothesized that 2NDEP could couple with PAMs through the AA site. This hypothesis was tested with the a7 mutant C190A, which is not activated by orthosteric agonists but is effectively activated by GAT107. The results showed that 2NDEP acts as an allosteric agonist of a7C190A when coapplied with the PAM PNU-120596. Also, the allosteric activity was nearly abolished upon coapplication with the AA site-selective antagonist 2,3,5,6MP-TQS (cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), consistent with AA site involvement. Overall, our findings show a novel mode of agonism through an allosteric site in the extracellular domain of a7 nAChR.

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators

“…TQS has proven to be a valuable scaffold for the development of analogs with diverse properties (Gill-Thind et al, 2015;Horenstein et al, 2016), including 4-(4-bromophenyl) (4BP)-TQS (Gill et al, 2011) and its active isomer, GAT107 (Papke et al, 2014). While typical PAMs require coapplication with an orthosteric agonist, allosteric activating positive allosteric modulators (ago-PAMs) appear to work at two sites, the transmembrane site controlled by the a7M159 residue and an allosteric activation site in the extracellular domain (Horenstein et al, 2016;Gulsevin et al, 2019). We evaluated the activity of the ago-PAM GAT107 on cells expressing a4 and b2L159M (Fig.…”

The allosteric activation of α7 nAChR by α-conotoxin MrIC is modified by mutations at the vestibular site

“…We first docked MrIA, MrIB, and MrIC into the ECD AA site of α7 nAChR and tested whether the three molecules could bind to this site. The ECD AA site ligands investigated so far in the literature interact with this site by burying their hydrophobic parts deep into the cavity defined by the vestibular loop residues 87-93 and 98-106 (21,(25)(26)(27). The bottom of the cavity is formed by the α7 nAChR residues F33, L56, Q57, M58, I90, and L91.…”

“…However, a vestibular binding site in the extracellular domain (ECD) named the "allosteric activation" (AA) site has been proposed to follow an alternative α7 nAChR activation mechanism (Figure 1). According to this hypothesis, the ligands that bind to the AA site can couple with the TMD directly and induce channel opening in the presence of a type II PAM (24)(25)(26). The MrIC activity profile is also consistent with that of allosteric agonists (17,19).…”

“…α7 nAChR mutants such as C190A are called "non orthosterically activatible receptors" (NOARs) because they cannot be activated by traditional agonists of the α7 nAChR, including ACh. Although NOARs cannot be activated orthosterically, they can be activated by standalone applications of agonist-PAMs (ago-PAMs) or allosteric agonists plus type II PAMs (21,25,31). Because α7C190A can only be activated allosterically, we tested MrIC responses with this mutant to provide further support for the allosteric activation hypothesis for MrIC.…”

“…These three peptides were previously synthesized and tested for activity at α7 nAChR upon co-application with a type II positive allosteric modulator (PAM) (17,19). Type II PAMs do not induce a response when applied by themselves, but they recover α7 nAChR from its desensitized Ds state, resulting in large responses to orthosteric and allosteric agonists (20,21). Of the three peptide molecules tested, MrIA had weak partial agonist activity and MrIB was found to have no effect.…”