title: Ligand-directed delivery of fluorophores to track native calcium-permeable AMPA receptors in neuronal cultures.
abstract: Subcellular trafficking of neuronal receptors is known to play a key role in synaptic development, homeostasis, and plasticity. We have developed a ligand-targeted and photo-cleavable probe for delivering a synthetic fluorophore to AMPA receptors natively expressed in neurons. After a receptor is bound to the ligand portion of the probe molecule, a proteinaceous nucleophile reacts with an electrophile on the probe, covalently bonding the two species. The ligand may then be removed by photolysis, returning the receptor to its non-liganded state while leaving intact the new covalent bond between the receptor and the fluorophore. This strategy was used to label polyamine-sensitive receptors, including calcium-permeable AMPA receptors, in live hippocampal neurons from rats. Here, we describe experiments where we examined specificity, competition, and concentration on labeling efficacy as well as quantified receptor trafficking. Pharmacological competition during the labeling step with either a competitive or non-competitive glutamate receptor antagonist prevented the majority of labeling observed without a blocker. In other experiments, labeled receptors were observed to alter their locations and we were able to track and quantify their movements. We used a small molecule, ligand-directed probe to deliver synthetic fluorophores to endogenously expressed glutamate receptors for the purpose of tracking these receptors on live, hippocampal neurons. We found that clusters of receptors appear to move at similar rates to previous studies. We also found that the polyamine toxin pharmacophore likely binds to receptors in addition to calcium-permeable AMPA receptors.
authors: Neuroscience and Behavior Program, University of Massachusetts, Amherst, Massachusetts, USA.
authors: Rosamund E
authors: RE
authors: Combs-Bachmann
authors: Jeffreys Nate
authors: JN
authors: Johnson
authors: Devaiah
authors: D
authors: Vytla
authors: Amanda M
authors: AM
authors: Hussey
authors: Maria L
authors: ML
authors: Kilfoil
authors: James J
authors: JJ
authors: Chambers
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D016503:Drug Delivery Systems / Q000379:methods*; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000378:metabolism*; D008024:Ligands; D008297:Male; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D018091:Receptors, AMPA / Q000032:analysis / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D018091:Receptors, AMPA; D002118:Calcium
keywords: AMPA receptors; calcium; fluorescent labeling; receptor trafficking