Abstract

Abstract ID 96289

Poster Board 125

Objective/Aim/Hypothesis: The objective of this work is to discover and characterize the first ligands able to identify selectively nicotinic acetylcholine receptors assembled from α7 and β2 subunits (α7β2-nAChR). Basal forebrain cholinergic neurons (BFCNs) express α7β2-nAChR. Elevated concentrations of oligomeric amyloid-β (associated with early Alzheimer’s disease) appear to mediate BFCN neuronal dysfunction. Expression of α7β2-nAChR by additional important cholinergic and GABAergic neuronal circuits of the central nervous system has been observed. However, further studies are stymied by a lack of ligands that can positively identify α7β2-nAChR from the related and more widespread α7-only homomeric nAChR subtype. This problem arises since α7-only- and α7β2-nAChR share identical agonist binding sites, located at α7/ α7 subunit interfaces, that are the targets of most current ligands. We hypothesized that new α-conotoxin (α-Ctx) ligands may instead selectively inhibit α7β2-nAChR via α7/β2 subunit interfaces.

Design/Approach/Methods: Two-electrode voltage clamp (TEVC) electrophysiology was used to screen ≫500 novel α-Ctxs for selectivity towards α7β2- over α7-only-nAChR functionally expressed in Xenopus oocytes. Kinetics analysis was used to probe the subtype selectivity and mechanism of α-Ctx antagonism. Molecular dynamics (MD) simulations were used to identify amino-acid residues at putative α7/β2 subunit α-Ctx binding sites. Site-directed mutagenesis was used to probe these hypothesized sites. TEVC electrophysiology was also used to determine α-Ctx activity at non- α7 nAChR subtypes.

Results: We discovered α-CtxPnIC analogs that selectively antagonize α7β2- over α7-only-nAChR. Kinetics analysis showed that association rates were similar across α-CtxPnIC analogs, and between α7β2- and α7-only-nAChR subtypes. Slower disassociation from α7β2- vs. α7-only-nAChR mainly drove selectivity towards α7β2-nAChR. The α-CtxPnIC [S4R] and [L10Y] analogs were the most selective towards α7β2-nAChR (18- and 57-fold vs. α7-only-nAChR, respectively). MD identified two sets of β2 subunit residues at the putative α7/β2 subunit interface for α-CtxPnIC analogs that differed from those at the known α7/ α7 subunit interface competitive agonist site. Mutating either set of β2 subunit residues to their α7 subunit equivalents partially reduced α-CtxPnIC [S4R] selectivity towards α7β2-nAChR. Activity of α-CtxPnIC analogs was generally low at non- α7-nAChR subtypes; data across analogs suggested approaches to decrease off-target affinity further.

Conclusions: We have identified the first ligands with selectivity towards α7β2-nAChR and proved a prototypical example of non-competitive antagonism by α-Ctxs. This discovery profoundly expands the scope of application of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). Further development of α-CtxPnIC analogs will enhance α7β2-nAChR selectivity, providing opportunities for basic and translational scientific breakthroughs related to nAChR biology, Alzheimer’s disease, and cholinergic contributions to cognition.

National Institutes of Health awards R01DA042749 (to P.W. and J.M.M.), R01DA043567 (to P.W. and W.I.), and R35GM136430 (to J.M.M.).

National Science Foundation award MCB-2111728 to W.I.

VCU Start-Up Funds (to P.W.).