Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors.
Alpha 7 subunit-containing nicotinic acetylcholine receptors (alpha7* nAChR) are involved in a variety of functions in the mammalian brain, including modulating neurotransmitter release and synaptic plasticity. Identifying the precise cellular distribution of alpha7* nAChRs with respect to the local neurochemical environment is crucial to understanding these biological roles. Current strategies for localising alpha7* nAChRs at the subcellular level have limitations. Anti-alpha7 subunit antibodies detect both assembled and unassembled subunits whereas biotinylated alphabungarotoxin (alphaBgt) only binds to assembled alpha7* nAChRs, but interpretation of labelling is marred by co-detection of endogenous tissue biotin. To overcome these problems, we have characterised a novel 1.4 nm gold alphaBgt conjugate used to directly localise alpha7* nAChR. Gold conjugation does not significantly decrease binding affinity, and gold alphaBgt specifically labels alpha7* nAChR in both unfixed and aldehyde-fixed tissue at the light and electron microscope levels, labelling being abolished in the presence of excess competing toxin. At the ultrastructural level, gold alphaBgt is associated with neuronal membranes and located at axon-dendritic synapses in the rat hippocampus CA1 stratum radiatum. These results reveal gold alphaBgt to be a valuable new tool in elucidating the functional neuroanatomy of alpha7* nAChR in the central nervous system.
Jones
Ian W
IW
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. i.w.jones@bath.ac.uk
Barik
Jacques
J
O'Neill
Michael J
MJ
Wonnacott
Susan
S
D000818:Animals; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D002038:Bungarotoxins / Q000032:analysis* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006046:Gold / Q000032:analysis* / Q000378:metabolism; D008297:Male; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000032:analysis* / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579165:Chrna7 protein, rat; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D006046:Gold