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 noncompetitive 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.

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

“…Competition experiments using NAS (1‐Naphthylacetyl spermine) and DNQX (6,7‐dinitroquinoxaline‐2,3‐dione) resulted in a marked decrease in the fluorescence signal. Such observations could not rule out tagging of other DNQX‐sensitive receptors e. g. kainate receptors [65] . Nonetheless, the results showcased localization and tracking of native iGluRs using a spinning disc confocal microscope.…”

“…Such observations could not rule out tagging of other DNQX-sensitive receptors e. g. kainate receptors. [65] Nonetheless, the results showcased localization and tracking of native iGluRs using a spinning disc confocal microscope.…”

“…Both subtypes play primary roles in the complex network of the Scheme 38. In blue scaffolds of alprenolol (64), propranolol (65) and pindolol (66). On the other side, linkers and fluorophore structures.…”

Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation

“…57 An electrophilic moiety on the probe, which contains the Cy3 fluorophore, forms a covalent bond with the receptor, in which the ligand probe can be photocleaved, freeing up the receptor to allow it to be functionally active. 57,58 In our current study, in the normoxia and hypoxia conditions there was a small fluorescent staining of cp-AMPARs in the RGCs maintained in RGC medium at 4 hours (Fig. 6A).…”

“…In the present experiment, Nanoprobe 1 was not photocleaved and, therefore, cp-AMPARs became endocytosed in the RGCs (Fig. 6A), due to blockage of the AMPAR channel pore, 58 which contributed some staining inside the cytosol. In this current study, AMPAR trafficking was not studied, just the expression of cp-AMPARs at the time following 4 hours of GD and OGD incubation.…”