The Novel α7β2-Nicotinic Acetylcholine Receptor Subtype Is Expressed in Mouse and Human Basal Forebrain: Biochemical and Pharmacological Characterization

Abstract

We examined a 7 b 2 -nicotinic acetylcholine receptor (a 7 b 2 -nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric a 7 -nAChRs and a 7 b 2 -nAChRs. a-Bungarotoxin affinity purification or immunoprecipitation with anti-a 7 subunit antibodies (Abs) was used to isolate nAChRs containing a 7 subunits from mouse or human brain samples. a 7 b 2 -nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using b 2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (a 7 ) 5 -, (a 7 ) 4 (b 2 ) 1 -, and (a 7 ) 3 (b 2 ) 2 -nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (a 7 ) 5 -nAChRs or for homomeric a 7 -nAChRs constituted from unlinked a 7 subunits. Pharmacological profiles were similar for (a 7 ) 5 -, (a 7 ) 4 (b 2 ) 1 -, and (a 7 ) 3 (b 2 ) 2 -nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at a 7 b 2 -versus a 7 -nAChRs. This study represents the first direct confirmation of a 7 b 2 -nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of a 7 b 2 -nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that a 7 b 2 -nAChR subunit isoforms with different a 7 /b 2 subunit ratios have similar pharmacological profiles to each other and to a 7 homopentameric nAChRs. This supports the hypothesis that a 7 b 2 -nAChR agonist activation predominantly or entirely reflects binding to a 7 /a 7 subunit interface sites.

α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

“…If b2 subunits are present in human adrenal chromaffin cell a3b4* nAChRs, they are likely to be restricted to the fifth or auxiliary position because no pharmacological evidence for an a3-b2 ligandbinding site was found. Additionally, b2 subunits may assemble with a7 subunits to form a7b2 nAChRs, which have recently been identified in rodent and human basal forebrain as well as mouse hippocampal interneurons (Liu et al, 2009b(Liu et al, , 2012Moretti et al, 2014).…”

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

“…Value of Using Tethered nAChR Pentamers and nAChR Cell Surface Binding Assays-The concatenated nAChR subunit approach offers substantial advantages in terms of expressing completely defined nAChR subtypes, including difficult-tocombine subunits (39,40,44). It also allows site-directed mutagenesis to be targeted to particular subunits when multiple copies are present in a pentameric complex (35)(36)(37).…”

“…Two-electrode Voltage Clamp Electrophysiology-Methodology for obtaining CRCs and maximal current responses from nAChR-injected oocytes has been published elsewhere (36,39,40). Briefly, at least 6 days post-injection, oocytes were voltageclamped at Ϫ70 mV with an Axoclamp 900A amplifier (Molecular Devices, Sunnyvale, CA).…”

Understanding the Bases of Function and Modulation of α7 Nicotinic Receptors: Implications for Drug Discovery

“…As shown in the figure, in a7, this region contains determinants of channel conductance (Andersen et al, , 2013 and mediates several intracellular signals (Paulo et al, 2009;Jones et al, 2010). (Liu et al, 2009(Liu et al, , 2012Moretti et al, 2014;Thomsen et al, 2015;Zoli et al, 2015). Interestingly, a7b2 are highly sensitive to blockade by Ab1-42, suggesting that they may play a unique role in the neuropathology of Alzheimer disease (Liu et al, 2009).…”