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|Title:||EFFECT OF CHRONIC NICOTINE TREATMENT ON EXPRESSION AND FUNCTION OF THE ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR IN CRYOPRESERVED CORTICAL NEURONS|
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|Authors/Affiliations:||1 John Mielke*; 1 Cristina Cassone; 1 Tanya Comas; 2 Anthony Krantis; 1 Geoffrey Mealing; |
1 National Research Council of Canada, Institute for Biological Sciences, Ottawa, ON, Canada; 2 QBM Cell Sciences & University of Ottawa, ON, Canada
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|Content:||Objectives: The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel belonging to the same family that includes the GABAA and glycine receptors. Although the nAChR may be formed from a collection of eleven subunits, the α7 homopentamer is of particular interest due to its widespread distribution throughout the cortex and hippocampus, and its role in processes underlying attention and memory. Nicotine, a tobacco-derived alkaloid and the prototypic nAChR agonist, has been shown to increase the number of nAChRs, however, very little is known regarding how chronic nicotine may affect the functional role of the receptor by influencing surface density of the α7 subunit. As a result, this study explored how chronic nicotine application altered the protein levels, surface expression, and binding capacity of the α7 nAChR in dissociated cortical neurons.|
Materials & Methods: Cryopreserved, embryonic rat cortical neurons were thawed, diluted in serum-free growth medium, and plated in 12-well plates according to established protocols1. Following 14 days in vitro, cultures were exposed to varying concentrations of nicotine (NIC) for 7 days, after which cells were homogenised and levels of the α7 subunit protein assessed. The membrane-impermeable cross-linking reagent BS3 was applied to control and NIC-treated cultures (to remove surface proteins), after which differences within the intracellular pool were examined using immunoblotting. To assess receptor function, cells were treated with a biotin-conjugated form of the specific α7 nAChR antagonist alpha-bungarotoxin (BGT) followed by streptavidin-HRP, which allowed binding capacity to be measured colourimetrically.
Results: Chronic NIC (10, 100, 1000 μM, 7 days) caused a 15-20% decrease in α7 protein levels. Under basal conditions, the α7 subunit displayed a remarkable degree of intracellular expression (percent of total: 87%), which was not the case for the glutamatergic GluR2 subunit (46%). When NIC was chronically applied in such a manner that α7 levels were not altered (100 μM, 3 days), there was no effect upon the subunitís intracellular density; however, there was a 14% decrease in the internal expression of the GluR2 subunit. Application of the same NIC protocol caused a minor increase (percent of untreated: 13%) in BGT binding.
Conclusions: In contrast to previous reports, we found that chronic NIC did not increase α7 levels, and, when extended long enough, actually caused a decline. Although there was no effect of chronic NIC on the intracellular concentration of α7, there was a slight increase in BGT binding, which suggests that extended application of agonist influenced antagonist binding without affecting receptor surface expression. Future experiments will explore further the influence of NIC on BGT binding, and the mechanisms underlying the strong intracellular retention of the α7 subunit.
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