Alexa Fluor 546-Arib[V11L;V16A] Is A Potent Ligand For Selectively Labeling 7 Nicotinic Acetylcholine Receptors
The 7* (*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 7 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 7 ligand -bungarotoxin binds to 1* and 9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC50 1.8 nM) and selectively blocked 7 nAChRs but not 1* or 9* 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 7 but not cells expressing 32, 34, or 42 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR 7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging 7 nAChRs. Â© 2010 The Authors. Journal Compilation Â© 2010 International Society for Neurochemistry.
Journal of Neurochemistry
Digital Object Identifier (DOI)
Hone, Arik J.; Whiteaker, Paul; Mohn, Jesse L.; Jacob, Michele H.; and McIntosh, J. Michael, "Alexa Fluor 546-Arib[V11L;V16A] Is A Potent Ligand For Selectively Labeling 7 Nicotinic Acetylcholine Receptors" (2010). Neurobiology. 380.