Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors.
The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.
Hone
Arik J
AJ
Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, Utah 84112, USA.
Whiteaker
Paul
P
Mohn
Jesse L
JL
Jacob
Michele H
MH
McIntosh
J Michael
JM
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes; D011485:Protein Binding; D011806:Quinolinium Compounds / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
C481052:Alexa fluor 546; C579167:Chrna7 protein, human; C579166:Chrna7 protein, mouse; C579165:Chrna7 protein, rat; D020916:Conotoxins; D005456:Fluorescent Dyes; D008024:Ligands; D011806:Quinolinium Compounds; D011978:Receptors, Nicotinic; C532888:alpha-conotoxin IB, Leu(11), Asp(16)-; D064569:alpha7 Nicotinic Acetylcholine Receptor