Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors

Abstract

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate excitatory synaptic transmission and are dynamically regulated during synaptic plasticity in the CNS. The membrane trafficking of AMPA receptors to synapses is critical for the regulation of the efficacy of excitatory synaptic transmission. Direct imaging of AMPA receptors in various cell compartments is important to dissecting the regulation of distinct steps in receptor membrane trafficking. In this study, we have developed an approach for the imaging of receptor trafficking with subunits tagged with a 13-aa alpha-bungarotoxin (BTX)-binding site (BBS). The small polypeptide neurotoxin BTX has been used for decades to study the nicotinic acetylcholine receptor. Similar high-affinity ligands are rarely available for most receptors. Engineering the BBS tag into receptor subunits allowed the high-affinity binding of fluorescent, radioactive, and biotinylated BTX to the tagged receptor subunits. By using this approach, the total receptor expression, surface expression, internalization, and insertion of receptors into the plasma membrane could be visualized and quantified in fixed or live cells including cultured neurons. The BBS tag is a flexible approach for labeling membrane proteins and studying their dynamic trafficking.

Fig. 1.

Expression of BBS-tagged receptor subunits in HEK 293 cells. (A) Amino acid sequence of the BBS. (B) Design of the BBS-tagged receptor subunits. BBS and GFP were inserted into the N-terminal extracellular region of the receptor subunits. (C and D) Western blotting of BBS-tagged subunits in HEK 293 cells. Both the GFP-GluR2 and BBS-GFP-GluR2 constructs were detected with anti-GFP (C) and anti-GluR2-C (D) antibodies. (E) Surface labeling of cells expressing BBS-GFP-GluR2 or GFP-GluR2 with anti-GluR2-N antibody (αGluR2N) or rhodamine-BTX (Rh-BTX). GFP, GFP signal detected in the cells. Both BBS-GFP-GluR2 and GFP-GluR2 constructs were expressed on the cell surface, whereas only the BBS-GFP-GluR2 construct was labeled with rhodamine-BTX on the cell surface.

Fig. 2.

Detection of internalization and insertion of BBS-tagged GluR2 in HEK 293 cells. (A) Cells transfected with BBS-GFP-GluR2 were incubated with rhodamine-BTX at 17°C for 30 min, washed to remove free rhodamine-BTX, incubated for several hours at 17°C (to inhibit internalization), and imaged at the time points indicated (0–6 h). (B) Quantification of the surface signals in the cells. BTX binding to the BBS-tagged GluR2 was stable for 6 h(n = 4). (C) Observation of internalization of BBS-GFP-GluR2 in the transfected HEK 293 cells. Cells transfected with BBS-GFP-GluR2 were incubated with rhodamine-BTX at 17°C for 30 min, washed to remove free rhodamine-BTX, incubated at 37°C to allow internalization of the receptor, and imaged at the indicated times (0–30 min) with confocal microscopy. Internalized BBS-GFP-GluR2 vesicles were seen in the intracellular region of the cells after incubation at 37°C. (D) Observation of plasma membrane insertion of BBS-GFP-GluR2 in transfected HEK 293 cells. Cells transfected with BBS-GFP-GluR2 were preincubated with unlabeled BTX at 17°C and incubated with rhodamine-BTX at 37°C for the indicated times to image the insertion of new receptors into the plasma membrane. Rapid insertion of the receptors into the plasma membrane within 10 min could be visualized with this method.

Fig. 3.

Detection of surface-expressed BBS-tagged GluR2 with rhodamine-BTX in cultured cortical and hippocampal neurons. (A) Cortical cultured neurons transfected with BBS-GFP-GluR2 were incubated with rhodamine-BTX at 17°C for 15 min, washed, and fixed. The rhodamine-BTX (Rh-BTX) labeled surface receptors, whereas the GFP signal showed the total expression of the BBS-GFP-GluR2 construct. (Scale bar: 10 μm.) (B) BBS-GFP-GluR2 was labeled with rhodamine-BTX on the surface of hippocampal neurons in culture. (C) GFP-GluR2 transfected neurons had a low background level of rhodamine-BTX binding.

Fig. 4.

Insertion and internalization of BBS-GFP-GluR2 in cortical neurons. Insertion of BBS-GFP-GluR2 into the surface membrane in the transfected neurons was observed by preincubating neurons with unlabeled BTX, followed by labeling with rhodamine-BTX (A and B). Neurons were preincubated for 15 min with unlabeled BTX, washed, incubated for 0 min (A) or 5 min (B) at 37°C, and labeled with rhodamine-BTX at for 15 min at 17°C. (Scale bar: 10 μm.) Magnified images of dendritic regions are shown in Insets. The insertion of new receptors could be seen on the cell surface in B. (C) Neurons were transfected with BBS-GFP-GluR2 and then labeled with rhodamine-BTX for 15 min at 17°C to label all cell-surface receptors. (D) Internalization of the BBS-GFP-GluR2 construct with transferrin in cortical neurons. The transfected neurons were observed after simultaneous labeling with rhodamine-BTX and FITC-transferrin. The BBS-GFP-GluR2 was partially colocalized with transferrin (arrows).

Fig. 5.

Time-lapse imaging of receptor insertion into the surface plasma membrane in cortical neurons. (A) The insertion of BBS-GFP-GluR2 into the surface membrane in the transfected neurons was observed after preincubating neurons with unlabeled BTX, followed by labeling with rhodamine-BTX. The clusters of receptors newly inserted in the surface could be seen in the dendrites and spines. Arrowheads show individual clusters of BBS-GFP-GluR2. (B) Quantification of signal intensity of individual clusters (n = 19).

Fig. 6.

BTX binding assay using ¹²⁵I-BTX in HEK 293 cell. (A) To measure surface expression, the cells expressing BBS-GFP-GluR2 or GFP-GluR2 were incubated with ¹²⁵I-BTX in the absence or presence of unlabeled BTX at 17°C for 30 min, the cells were washed, and the ¹²⁵I-BTX bound in the cell surface was measured. Robust ¹²⁵I-BTX binding was detected in BBS-GFP-GluR2-transfected cells. (B) To measure total expression, detergent extracts of membrane fractions were incubated with ¹²⁵I-BTX with or without unlabeled BTX for 30 min at room temperature, spotted onto DEAE filters, washed, and counted. (C) The time course of ¹²⁵I-BTX binding was measured by using detergent extracts of membrane fractions for the indicated times. ¹²⁵I-BTX binding was completed within 1 min. (D) The detergent extracts of membrane fractions were incubated with various concentrations of ¹²⁵I-BTX, and the dissociation constant (K_d affinity) was quantified (K_d = 1.36 × 10^–8 M) from the resulting Scatchard plot (Inset). (E) The BBS-GFP-GluR2 was specifically detected on Western blot by using biotinylated BTX and streptavidin-HRP. Lanes show untransfected cells (unTF), vector, GFP-GluR2, and BBS-GFP-GluR2 from left to right. (F) Surface BBS-GFP-GluR2, but not GFP-GluR2, was isolated from the transfected cells by using biotinylated BTX and NeutrAvidin beads.