Cellular distribution of the nicotinic acetylcholine receptor alpha7 subunit in rat hippocampus.
The hippocampus is a region of the mammalian brain that has been extensively studied due to its role in many forms of memory. To better understand hippocampal function, significant attention has focused upon the cellular distribution of ligand-gated ion channels. Despite strong cholinergic innervation from the basal forebrain and a dense expression of nicotinic acetylchoine receptors (nAChRs), the cellular distribution of subunits forming these receptors has received little attention. We used organotypic hippocampal slice cultures (OHSCs) to study native alpha7 subunits, which, unlike other nAChR subunits, form a homomeric receptor. Cell-surface biotinylation, cross-linking of surface proteins, and sub-cellular fractionation all revealed a very limited presence of the subunit at the plasma membrane. In contrast, subunits of other receptors displayed significant surface expression. Notably, subunits in adult hippocampal tissue were distributed in a fashion similar to that observed in OHSCs. To monitor alpha7 subunits contained in functional nAChRs, a colourimetric assay using alpha-bungarotoxin (a specific alpha7 nAChR antagonist) was developed, and revealed a majority of binding at the cell surface. To change alpha7 subunit distribution, OHSCs were treated with compounds known to affect other ionotropic receptors-insulin, genistein, and elevated external K(+); however, neither subunit surface expression nor antagonist binding was affected. Our data reveal that hippocampal neurons possess a large internal population of alpha7 subunits under basal conditions, which persists during stimuli affecting tyrosine phosphorylation or neuronal activity. The nature of the internal pool of alpha7 subunits remains to be determined, but should have important implications for hippocampal activity.
Mielke
John G
JG
Department of Health Studies and Gerontology, Faculty of Applied Health Sciences, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1. jgmielke@uwaterloo.ca
Mealing
Geoffrey A R
GA
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000502:physiology; D001681:Biological Assay / Q000379:methods; D002038:Bungarotoxins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology; D006624:Hippocampus / Q000378:metabolism* / Q000648:ultrastructure; D009448:Neurochemistry / Q000379:methods; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579165:Chrna7 protein, rat; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D014443:Tyrosine; D000109:Acetylcholine; D011188:Potassium