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The novel ?7?2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization.

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

SUBMITTER: Moretti M

PROVIDER: S-EPMC4152907 | biostudies-other | 2014 Sep

REPOSITORIES: biostudies-other

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The novel α7β2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization.

Moretti Milena M Zoli Michele M George Andrew A AA Lukas Ronald J RJ Pistillo Francesco F Maskos Uwe U Whiteaker Paul P Gotti Cecilia C

Molecular pharmacology 20140707 3

We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subun ...[more]

PMID: 25002271

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Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

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The novel ?7?2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization.

Publications

The novel α7β2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization.

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