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Chemical labelling for visualizing native AMPA receptors in live neurons

View Article: PubMed Central - PubMed

ABSTRACT

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

No MeSH data available.


Chemical labelling of AMPARs using CAM reagents.(a) Schematic illustration of chemical labelling of AMPARs driven by the selective ligand-protein recognition using CAM reagents. Lg, ligand moiety; Nu, nucleophilic amino acid residue. (b) Chemical structures of the CAM reagents. The detailed chemical structures are shown in Supplementary Methods.
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f1: Chemical labelling of AMPARs using CAM reagents.(a) Schematic illustration of chemical labelling of AMPARs driven by the selective ligand-protein recognition using CAM reagents. Lg, ligand moiety; Nu, nucleophilic amino acid residue. (b) Chemical structures of the CAM reagents. The detailed chemical structures are shown in Supplementary Methods.

Mentions: For covalent attachment of small chemical probes to AMPARs, we applied ligand-directed acyl imidazole (LDAI) chemistry, a traceless protein labelling method1920. LDAI-based chemical labelling is driven by selective ligand-protein recognition, which facilitates an acyl substitution reaction of labelling reagents to nucleophilic amino acid residues (Lys, Ser or Tyr) located near the ligand-binding domain (Fig. 1a). Here we carefully designed labelling reagents for AMPARs by taking into consideration the selectivity of the affinity ligand, the orientation of the acyl imidazole group, and the total charges of the labelling reagents. We chose 6-pyrrolyl-7-trifluoromethyl-quinoxaline-2,3-dione (PFQX) as a ligand, because PFQX exhibits a sufficient affinity (Ki value of 170 nM) and selectivity for AMPARs over other glutamate receptors, including N-methyl-D-aspartate (NMDA) and kainate receptors3435 (Fig. 1b). In addition, this negatively charged ligand is relatively hydrophilic, which offers the possibility of selective labelling of cell-surface AMPARs by suppressing permeation of the labelling reagents into live neurons. The pyrrolyl moiety of PFQX was assumed to be accessible to the surface of the ligand-binding domain based on X-ray structural analysis of an AMPAR bound with ZK200775, an antagonist similar to PFQX36. Thus, a variety of probes were connected to this pyrrolyl moiety of PFQX via a reactive acyl imidazole linker. We prepared labelling reagents with various spacers between the reactive acyl imidazole unit and the ligand to finely control the position of the acyl imidazole unit on the AMPAR surface, and we termed this series of probes ‘chemical AMPAR modification' (CAM) reagents (Fig. 1b). Notably, the labelling procedure using these reagents is very simple, involving only brief incubation and washing procedures. In addition, the excess labelling reagents and the cleaved ligand moiety can be readily washed out (Fig. 1a).


Chemical labelling for visualizing native AMPA receptors in live neurons
Chemical labelling of AMPARs using CAM reagents.(a) Schematic illustration of chemical labelling of AMPARs driven by the selective ligand-protein recognition using CAM reagents. Lg, ligand moiety; Nu, nucleophilic amino acid residue. (b) Chemical structures of the CAM reagents. The detailed chemical structures are shown in Supplementary Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385570&req=5

f1: Chemical labelling of AMPARs using CAM reagents.(a) Schematic illustration of chemical labelling of AMPARs driven by the selective ligand-protein recognition using CAM reagents. Lg, ligand moiety; Nu, nucleophilic amino acid residue. (b) Chemical structures of the CAM reagents. The detailed chemical structures are shown in Supplementary Methods.
Mentions: For covalent attachment of small chemical probes to AMPARs, we applied ligand-directed acyl imidazole (LDAI) chemistry, a traceless protein labelling method1920. LDAI-based chemical labelling is driven by selective ligand-protein recognition, which facilitates an acyl substitution reaction of labelling reagents to nucleophilic amino acid residues (Lys, Ser or Tyr) located near the ligand-binding domain (Fig. 1a). Here we carefully designed labelling reagents for AMPARs by taking into consideration the selectivity of the affinity ligand, the orientation of the acyl imidazole group, and the total charges of the labelling reagents. We chose 6-pyrrolyl-7-trifluoromethyl-quinoxaline-2,3-dione (PFQX) as a ligand, because PFQX exhibits a sufficient affinity (Ki value of 170 nM) and selectivity for AMPARs over other glutamate receptors, including N-methyl-D-aspartate (NMDA) and kainate receptors3435 (Fig. 1b). In addition, this negatively charged ligand is relatively hydrophilic, which offers the possibility of selective labelling of cell-surface AMPARs by suppressing permeation of the labelling reagents into live neurons. The pyrrolyl moiety of PFQX was assumed to be accessible to the surface of the ligand-binding domain based on X-ray structural analysis of an AMPAR bound with ZK200775, an antagonist similar to PFQX36. Thus, a variety of probes were connected to this pyrrolyl moiety of PFQX via a reactive acyl imidazole linker. We prepared labelling reagents with various spacers between the reactive acyl imidazole unit and the ligand to finely control the position of the acyl imidazole unit on the AMPAR surface, and we termed this series of probes ‘chemical AMPAR modification' (CAM) reagents (Fig. 1b). Notably, the labelling procedure using these reagents is very simple, involving only brief incubation and washing procedures. In addition, the excess labelling reagents and the cleaved ligand moiety can be readily washed out (Fig. 1a).

View Article: PubMed Central - PubMed

ABSTRACT

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

No MeSH data available.