ABSTRACT

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2pHFAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2pHFAP GABAARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2pHFAP GABAARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2pHFAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2pHFAP–MG dye approach reveals enhanced GABAAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP–MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAAR trafficking.

INTRODUCTION

γ-Aminobutyric acid (GABA) type A receptors (GABAARs) are ligand-gated ionotropic Cl channels responsible for most fast inhibitory neurotransmission in the adult central nervous system (CNS). This inhibition requires binding of the neurotransmitter GABA, which induces ion channel opening, Cl influx and subsequent membrane hyperpolarization. The majority of GABAARs are composed of two α subunits, two β subunits and a γ2 subunit forming a heteropentamer, but considerable subunit heterogeneity can exist (α1–6, β1–3, γ1–3, δ, ɛ, θ, π, ρ1–3) (Luscher et al., 2011). The γ2 subunit (encoded by GABRG2) plays a critical role in GABAAR function, as it is necessary for receptor synaptic targeting and cluster maintenance (Alldred et al., 2005; Essrich et al., 1998; Rovo et al., 2014; Schweizer, 2003; Sumegi et al., 2012), coassembles with nearly all α and β subunits (Möhler et al., 2001), and is the only subunit characterized to undergo ubiquitylation leading to lysosomal degradation (Arancibia-Carcamo et al., 2009). The degree of γ2-mediated GABAAR synaptic clustering directly impacts the strength of GABAergic synaptic inhibition and is dynamically regulated by changes in receptor trafficking (Jacob et al., 2008; Luscher et al., 2011). A number of endogenous and pharmacological agents including GABA, neurosteroids, ethanol and benzodiazepines are known to influence receptor trafficking (Calkin and Barnes, 1994; Carver and Reddy, 2013; Hablitz et al., 1989; Kumar et al., 2010; Liang et al., 2007; Shen et al., 2011; Tehrani and Barnes, 1988, 1991; Wan et al., 1997). Despite this knowledge, precise GABAAR trafficking mechanisms induced by these and other clinically relevant compounds remain underexplored at the molecular level.

Real-time receptor trafficking measurements typically rely on genetically encoded fluorophores to track single protein localization and movement within living cells. In contrast to immunofluorescence-based live-cell techniques, which require reliable antibodies and are restricted to measurements of surface proteins (Arancibia-Carcamo et al., 2006), fluorescent tags allow for identification of a protein from synthesis through degradation. Despite this advantage, traditional fluorophores still remain limited in their ability to spatially resolve surface from internal populations without the use of total internal reflection fluorescence (TIRF)-imaging approaches (Kirchhausen, 2009). This issue led to the generation of pH-sensitive fluorescent proteins, such as pHluorin (pHGFP), which exhibit fluorescence in extracellular environments with alkaline pH (pH ∼7.4), but not in more acidic areas like intracellular vesicles (Sankaranarayanan et al., 2000). However, pHGFP is not without limitations as some discernible signal can be observed in the endoplasmic reticulum (ER) where pH can be roughly 7.2 (Asokan and Cho, 2002). One possible way to resolve protein trafficking occurring at the cell surface from the intracellular space is to use compartment-specific high-affinity labeling techniques. We previously described a labeling method utilizing a GABAAR subunit genetically tagged with an α-bungarotoxin-binding site, which selectively binds cell-excluded fluorescent bungarotoxin, allowing for selective monitoring of receptor insertion and internalization (Brady et al., 2014). Unfortunately, the inherent fluorescence of Alexa Fluor dye-coupled bungarotoxins necessitates extensive washing after labeling to reduce background signal. Furthermore, bungarotoxins were recently shown to function as antagonists of the GABAAR, further complicating the use of this reagent (Hannan et al., 2015). To overcome these obstacles associated with GABAAR imaging, we have employed an innovative paired optical reporter system where two individually non-fluorescent components become highly fluorescent upon binding: a genetically encoded fluorogen-activating peptide (FAP tag) and exogenously applied Malachite Green (MG) dyes. Multiple FAPs are described as antibody single-chain variable fragments (scFvs) that selectively bind MG synthetic dyes with high specificity and affinity (Szent-Gyorgyi et al., 2008). These synthetic dyes are non-fluorescent in solution until bound by their respective FAP and can be modified to have distinct characteristics including cell permeability, pH-sensitivity and various fluorescence properties (Fisher et al., 2014; Grover et al., 2012; Perkins et al., 2017 preprint; Saunders et al., 2012; Zhang et al., 2015). The FAP–dye system offers many advantages for live-imaging: (i) the dyes can be added directly to a culture dish and saturate the target FAP in seconds; (ii) a number of distinct dyes can be used for the same genetically encoded FAP; and (iii) the dyes are highly specific for their target FAP. Moreover, high-affinity MG–FAP binding means that a stable fluorescent module that allows for measurements of receptors undergoing internalization and recycling is formed (Pratt et al., 2015; Szent-Gyorgyi et al., 2013; Yan et al., 2015). Measuring drug-induced changes in FAP-tagged receptor trafficking has proven largely successful (Fisher et al., 2010, 2014; Grover et al., 2012; Pratt et al., 2015; Snyder et al., 2015; Wu et al., 2014) placing this technique at the forefront of pharmacological screening.

In order to design a tool to track synaptic GABAAR internalization and trafficking, we engineered a γ2 subunit encoding a pHGFP and the fluorogen-activating peptide dl5 (γ2pHFAP) (He et al., 2016; Saunders et al., 2013; Szent-Gyorgyi et al., 2008, 2013). We find that γ2pHFAP-containing GABAARs are trafficked to the cell surface in both HEK293 cells and primary neurons, and are localized appropriately at synapses. We further demonstrate how this construct can be combined with MG dye derivatives to measure alterations in surface localization and intracellular trafficking through high-resolution confocal microscopy approaches. Finally, using these FAP-based methods, we found that an in vitro seizure model induced rapid loss of dye-labeled synaptic GABAARs concomitant with enhanced targeting of internalized receptors to lysosomal compartments, key results that were not detectable when using the pHGFP signal alone. We therefore demonstrate an innovative tool to monitor multistage synaptic GABAAR trafficking.

RESULTS

The dual reporter γ2pHFAP is expressed in HEK293 cells

We began by introducing the dl5 FAP into a previously characterized N-terminal pHGFP γ2-construct (γ2pHGFP) that functions comparably to wild-type γ2 (Jacob et al., 2005; Kittler et al., 2000b; Muir et al., 2010; Renner et al., 2012) (Fig. 1A). The FAP will selectively bind MG dyes with distinct biophysical and fluorescence properties. For example, the MG derivative 2-({4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutanoyl}amino)ethanesulfonate (MG-BTau) is cell impermeant, binds to dl5 FAP with a high binding affinity (Kd value less than 0.5 nM), and is non-fluorescent until dl5 FAP binding occurs, which causes activation of fluorescence in the far-red spectral region (680 nm) (Larsen et al., 2016; Pratt et al., 2015; Szent-Gyorgyi et al., 2013; Yan et al., 2015) (Fig. 1B). We first examined whether the full-length γ2pHFAP construct was appropriately expressed in HEK293 cells. GABAAR β3 subunits (encoded by GABRB3) were co-expressed in these experiments as a β subunit is required for trafficking of receptors to the cell surface (Connolly et al., 1996). Western blot analysis of non-transfected, γ2pHGFP control and γ2pHFAP-expressing cells reveal γ2pHFAP is ∼25 kDa larger than γ2pHGFP, consistent with the molecular mass of the dl5 FAP (Szent-Gyorgyi et al., 2013) (Fig. 1C). Next, we evaluated surface expression and specificity of MG-BTau dye labeling of GABAARs in cells transiently transfected with β3+γ2pHFAP or β3+γ2pHGFP subunits. Addition of MG-BTau to a live-cell culture should swiftly and selectively label surface GABAARs containing γ2pHFAP. To test this dye-based labeling approach, living cells plated on glass-bottom Mattek dishes were pulse-labeled with 250 nM MG-BTau in HEPES-buffered saline (HBS) for 1 min at room temperature, washed five times with saline, and then immediately imaged by confocal microscopy. We found that HEK293 cells expressing γ2pHFAP demonstrate selective receptor surface labeling by MG-BTau, while γ2pHGFP control cells show no cell membrane MG-BTau fluorescence (although occasional low-level background MG-BTau binding can occur with cellular debris; Yan et al., 2015) (Fig. 1D). The majority of cells express heteromeric receptors composed of β3+γ2 subunits, although a few cells express the γ2 subunit alone, leading to subunit retention in the ER and no MG-BTau signal in γ2pHFAP cells (Fig. 1D, yellow arrowheads). These results highlight two key advantages of FAP–fluorogen labeling over pHGFP alone: (1) a higher signal-to-noise ratio, and (2) the removal of pHGFP intracellular background fluorescence generated by ER-localized receptors where pH can be roughly 7.2 (Asokan and Cho, 2002).

Fig. 1.

The novel optical GABAAR paired reporter system. (A) The fluorogen-activating peptide (FAP) dl5 was inserted upstream of an N-terminal pH-sensitive GFP tag (pHGFP) in a γ2 subunit construct. Cartoon schematic of γ2pHFAP subunit. (B) The MG dye, MG-BTau, is cell impermeant and ‘dark’ in solution. Fluorescence is generated when MG dye binds to surface receptors containing the γ2pHFAP subunit. (C,D) HEK293 cells were transfected with β3+γ2pHGFP control or β3+γ2pHFAP (β3 subunits are needed to form surface-targeted receptors). (C) Representative western blot from non-transfected (NT), β3+γ2pHGFP control and β3+γ2pHFAP expressing cells (n=2 experiments). (D) HEK293 cells transfected with β3+γ2pHGFP control or β3+γ2pHFAP were pulse-labeled with 250 nM MG-BTau dye for 1 min prior to live-cell imaging. pHGFP fluorescence is shown in green and MG-BTau is shown in blue in the Merge panels. Boxed areas are enlarged in lower images. The MG-BTau signal is selectively localized at the cell surface in γ2pHFAP-expressing cells and is absent in γ2pHGFP control cells. Note that with double transient transfection (β3+γ2pHFAP), some cells contain the γ2pHFAP subunit alone (yellow arrowheads), leading to intracellular retention and a pHGFP signal (green) but no MG dye labeling (blue). Scale bars: 20 μm.

Fig. 1.

The novel optical GABAAR paired reporter system. (A) The fluorogen-activating peptide (FAP) dl5 was inserted upstream of an N-terminal pH-sensitive GFP tag (pHGFP) in a γ2 subunit construct. Cartoon schematic of γ2pHFAP subunit. (B) The MG dye, MG-BTau, is cell impermeant and ‘dark’ in solution. Fluorescence is generated when MG dye binds to surface receptors containing the γ2pHFAP subunit. (C,D) HEK293 cells were transfected with β3+γ2pHGFP control or β3+γ2pHFAP (β3 subunits are needed to form surface-targeted receptors). (C) Representative western blot from non-transfected (NT), β3+γ2pHGFP control and β3+γ2pHFAP expressing cells (n=2 experiments). (D) HEK293 cells transfected with β3+γ2pHGFP control or β3+γ2pHFAP were pulse-labeled with 250 nM MG-BTau dye for 1 min prior to live-cell imaging. pHGFP fluorescence is shown in green and MG-BTau is shown in blue in the Merge panels. Boxed areas are enlarged in lower images. The MG-BTau signal is selectively localized at the cell surface in γ2pHFAP-expressing cells and is absent in γ2pHGFP control cells. Note that with double transient transfection (β3+γ2pHFAP), some cells contain the γ2pHFAP subunit alone (yellow arrowheads), leading to intracellular retention and a pHGFP signal (green) but no MG dye labeling (blue). Scale bars: 20 μm.

γ2pHFAP does not disrupt GABAAR channel function

We further investigated the ability of γ2pHFAP to assemble into functional heteromeric α2β3γ2 GABAARs with normal Cl channel activity in response to the endogenous agonist GABA, which binds between the α and β subunits. Patch clamp recordings were used to determine the GABA dose–response curve for HEK293 cells expressing receptors composed of α2β3γ2pHGFP or α2β3γ2pHFAP. Concentration–response analysis revealed these receptors exhibit similar GABA EC50 values (α2β3γ2pHGFP, 22.0±6.2 µM; α2β3γ2pHFAP, 14.3±8.9 µM; P>0.05) and Hill coefficients (α2β3γ2pHGFP, 1.14±0.26; α2β3γ2pHFAP, 0.90±0.29; mean±s.e.m., P>0.05) (Fig. 2A). The EC50 values are consistent with previously reported values for receptors containing this subunit composition (Mortensen et al., 2011). To directly test whether addition of the FAP tag altered drug binding and functional properties of the γ2 subunit, we compared the potentiation response induced by the benzodiazepine diazepam (DZ), a positive GABAAR allosteric modulator that binds at the interface of γ2 and specific α subunits (α1, 2, 3 or 5) (Fig. 2B) (Möhler et al., 2001). We found no significant difference in the DZ potentiation of the EC20 GABA response between α2β3γ2pHGFP- and α2β3γ2pHFAP-expressing cells (Fig. 2C, fold DZ potentiation α2β3γ2pHGFP=3.1±0.5, α2β3γ2pHFAP=2.8±0.6; P>0.05). Finally, we tested whether binding of a MG dye to the γ2pHFAP subunit would interfere with receptor function. We compared the DZ potentiation response of γ2pHFAP before and after co-application of MG-BTau. Binding of MG-BTau to the receptors did not alter the response to DZ (Fig. 2D, fold DZ potentiation γ2pHFAP without dye=4.0±0.4, γ2pHFAP with dye=3.9±1.0; mean±s.e.m., P>0.05). Taken together, these results suggest that GABAARs incorporating γ2pHFAP maintain normal receptor function and responsiveness to GABA and DZ in the presence of MG dye.

Fig. 2.

Recombinant GABAARs containing γ2pHFAP maintain responsiveness to GABA and the benzodiazepine drug DZ in HEK293 cells. (A) The GABA dose–response curve in α2β3γ2pHGFP- and α2β3γ2pHFAP-expressing cells is equivalent. 1 s GABA applications were made at ≥120 s intervals and peak response was measured. Curves were fit with the Hill equation, and the EC50 was determined. (B) GABA currents are equivalently potentiated by 1 μM DZ (at the GABA EC20). Representative traces show responses to application of GABA (black) and of GABA with DZ (red). (C) Quantification of DZ potentiation (n=5–8 cells per treatment). (D) DZ potentiation of the GABA response in α2β3γ2pHFAP-expressing cells is not altered by the presence of 100 nM MG-BTau dye (n=4 cells per treatment). Data in C and D is presented as mean±s.e.m. Results are not significantly different (Student's t-tests).

Fig. 2.

Recombinant GABAARs containing γ2pHFAP maintain responsiveness to GABA and the benzodiazepine drug DZ in HEK293 cells. (A) The GABA dose–response curve in α2β3γ2pHGFP- and α2β3γ2pHFAP-expressing cells is equivalent. 1 s GABA applications were made at ≥120 s intervals and peak response was measured. Curves were fit with the Hill equation, and the EC50 was determined. (B) GABA currents are equivalently potentiated by 1 μM DZ (at the GABA EC20). Representative traces show responses to application of GABA (black) and of GABA with DZ (red). (C) Quantification of DZ potentiation (n=5–8 cells per treatment). (D) DZ potentiation of the GABA response in α2β3γ2pHFAP-expressing cells is not altered by the presence of 100 nM MG-BTau dye (n=4 cells per treatment). Data in C and D is presented as mean±s.e.m. Results are not significantly different (Student's t-tests).

Neurons incorporate γ2pHFAP into synaptic GABAergic clusters

To confirm that γ2pHFAP is fully expressed in cultured neurons, we compared non-transfected rat cortical neurons to those transfected with γ2pHFAP or γ2pHGFP control at plating (Fig. 3). Neurons were lysed 14 days after transfection and subsequently immunoblotted with anti-GFP antibody (Fig. 3A), revealing substantial expression of γ2pHFAP and γ2pHGFP, as seen in HEK293 cells (Fig. 1C). We then tested whether neurons with γ2pHFAP GABAARs could selectively bind and activate MG-BTau dye fluorescence. Cortical neurons at 12 days in vitro (DIV) expressing γ2pHGFP or γ2pHFAP were pulse-labeled with 250 nM MG-BTau dye for 1 min at room temperature, then immediately washed and used for live-cell imaging. Only γ2pHFAP-expressing neurons demonstrate dye activation, and this signal colocalizes with the surface synaptic clusters marked by pHGFP (Fig. 3B). Furthermore, it is evident that MG-BTau labeling in neurons also has a higher signal-to-noise ratio than pHGFP due to, first, the continually generated pHGFP signal of diffuse newly inserted extrasynaptic γ2pHFAP receptors that are not yet clustered at synapses, which contrasts with the small MG-BTau pulse-labeled extrasynaptic population (if the MG dye was continually present rather than being washed away, its extrasynaptic signal would be more similar to pHGFP) and, second, the low but observable pHGFP background from ER-resident γ2pHFAP subunits, whereas there is no ER signal from MG-BTau labeling. These data establish MG dyes as a selective label for synaptic γ2pHFAP-containing GABAAR populations in living primary cortical neurons.

Fig. 3.

γ2pHFAP is fully expressed in neurons and appropriately clustered at GABAergic synapses. (A) Detection of γ2pHGFP control and γ2pHFAP receptors via western blotting of lysates from cortical neurons 14 days post-transfection. NT, non-transfected. n=3 neuronal cultures. (B) DIV 12 neurons expressing γ2pHGFP control or γ2pHFAP were pulse-labeled with 250 nM MG-BTau dye for 1 min prior to live-cell imaging. Surface GABAARs are selectively labeled in γ2pHFAP neurons as evidenced by extensive colocalization of the pHGFP signal (green, Merge) and MG-BTau signal (blue, Merge). Note that in γ2pHGFP control neurons, surface GABAARs are not labeled by MG-BTau dye. Boxed areas enlarged in lower images. (C) Confocal fixed immunofluorescence images of γ2pHGFP control and γ2pHFAP-expressing neurons with presynaptic GABAergic terminals labeled with anti-VGAT antibody. pHGFP fluorescence (green in Merge) shows intracellular γ2 subunit and synaptically localized receptors on dendrites that are colocalized with VGAT (red in Merge). Boxed areas enlarged in the right-hand images. (D) VGAT and surface γ2-containing receptor synaptic clusters were quantified. Scatter plot graphs showing mean area and mean fluorescence intensity in neurons transfected with γ2pHGFP control or γ2pHFAP. No changes were observed in GABAergic presynaptic terminals as measured by VGAT area and intensity. Synaptic γ2 GABAAR measurements were determined from the pHGFP signal colocalized with VGAT: synaptic γ2 GABAAR fluorescence area was equivalent, while the intensity was slightly lower in γ2pHFAP neurons. n=54–57 neurons from three cultures for each condition; results are mean±95% confidence interval. ***P<0.001 (Student's t-test). Scale bars: 20 μm (main panels), 1 μm (enlargements).

Fig. 3.

γ2pHFAP is fully expressed in neurons and appropriately clustered at GABAergic synapses. (A) Detection of γ2pHGFP control and γ2pHFAP receptors via western blotting of lysates from cortical neurons 14 days post-transfection. NT, non-transfected. n=3 neuronal cultures. (B) DIV 12 neurons expressing γ2pHGFP control or γ2pHFAP were pulse-labeled with 250 nM MG-BTau dye for 1 min prior to live-cell imaging. Surface GABAARs are selectively labeled in γ2pHFAP neurons as evidenced by extensive colocalization of the pHGFP signal (green, Merge) and MG-BTau signal (blue, Merge). Note that in γ2pHGFP control neurons, surface GABAARs are not labeled by MG-BTau dye. Boxed areas enlarged in lower images. (C) Confocal fixed immunofluorescence images of γ2pHGFP control and γ2pHFAP-expressing neurons with presynaptic GABAergic terminals labeled with anti-VGAT antibody. pHGFP fluorescence (green in Merge) shows intracellular γ2 subunit and synaptically localized receptors on dendrites that are colocalized with VGAT (red in Merge). Boxed areas enlarged in the right-hand images. (D) VGAT and surface γ2-containing receptor synaptic clusters were quantified. Scatter plot graphs showing mean area and mean fluorescence intensity in neurons transfected with γ2pHGFP control or γ2pHFAP. No changes were observed in GABAergic presynaptic terminals as measured by VGAT area and intensity. Synaptic γ2 GABAAR measurements were determined from the pHGFP signal colocalized with VGAT: synaptic γ2 GABAAR fluorescence area was equivalent, while the intensity was slightly lower in γ2pHFAP neurons. n=54–57 neurons from three cultures for each condition; results are mean±95% confidence interval. ***P<0.001 (Student's t-test). Scale bars: 20 μm (main panels), 1 μm (enlargements).

Synapse formation and receptor clustering are critical for neuronal development and regulation of inhibitory neurotransmission. To determine whether γ2pHFAP constructs cluster normally at GABAergic synapses in mature neurons, we transfected cortical neurons at DIV 0 with γ2pHGFP control or γ2pHFAP and fixed the cells for immunofluorescence studies at DIV 15. We assessed receptor synaptic localization by measuring colocalization of the pHGFP-tagged receptors with the vesicular GABA transporter (VGAT), a presynaptic marker of GABAergic synapses (Fig. 3C). Expression of γ2pHFAP did not alter presynaptic GABAergic input as indicated by unchanged VGAT levels (intensity) and area compared to that in the control (Fig. 3D). Additionally, the size of postsynaptic GABAergic synapses was not significantly different between constructs, although the mean intensity of γ2pHGFP synapses was 20.7% (95% confidence interval) greater than the intensity of γ2pHFAP, likely due to higher overall expression of the control subunit. These results indicate that the full-length γ2pHFAP construct is expressed, assembles with endogenous subunits into receptors, traffics to synapses and does not disturb neuronal development.

Intracellular trafficking of internalized GABAARs can be tracked using γ2pHFAP

Having validated and established the application of FAP technology to generate a synaptic GABAAR reporter system, we first explored the experimental flexibility afforded by the distinct characteristics of available MG dyes to study receptor trafficking. First, we examined whether MG-BTau-labeled surface receptors could be identified in endosomal pathways following internalization. DIV 12–13 γ2pHFAP neurons were pulse-labeled with 100 nM MG-BTau, transferred to 10°C or 37°C HBS for 30 min, and were then fixed and immunostained for the early endosome marker EEA1 (Fig. 4A). Neurons maintained at 37°C demonstrated greater mean intensity and area of MG-BTau colocalized at EEA1 vesicles compared to those kept at 10°C to inhibit internalization, indicating MG-BTau labeling can also be used to track internalized GABAAR pools (Fig. 4B).

Fig. 4.

MG-BTau dye signal shows trafficking of γ2pHFAP GABAARs to early endosomes. (A) DIV 12 γ2pHFAP-expressing neurons were pulse-labeled with 100 nM MG-BTau dye for 2 min, then incubated in HBS at 37°C or 10°C for 30 min prior to fixation. MG-BTau-labeled neurons (blue) were permeabilized and stained for the early endosome marker EEA1 (red). pHGFP (green) is visible throughout the cell after fixation. The boxed area is enlarged in the right-hand panels [EEA1 (red), MG-BTau (blue) and Merge panels]. Yellow circles show colocalized EEA1 and MG-BTau signal. (B) The intensity and area of MG-BTau-labeled receptors colocalized with EEA1-positive intracellular vesicles is increased when endocytosis is not inhibited by 10°C incubation. n=25 neurons from three cultures for each condition; results are mean±s.e.m. *P≤0.05, **P<0.01 (Student's t-test). Scale bars: 5 μm.

Fig. 4.

MG-BTau dye signal shows trafficking of γ2pHFAP GABAARs to early endosomes. (A) DIV 12 γ2pHFAP-expressing neurons were pulse-labeled with 100 nM MG-BTau dye for 2 min, then incubated in HBS at 37°C or 10°C for 30 min prior to fixation. MG-BTau-labeled neurons (blue) were permeabilized and stained for the early endosome marker EEA1 (red). pHGFP (green) is visible throughout the cell after fixation. The boxed area is enlarged in the right-hand panels [EEA1 (red), MG-BTau (blue) and Merge panels]. Yellow circles show colocalized EEA1 and MG-BTau signal. (B) The intensity and area of MG-BTau-labeled receptors colocalized with EEA1-positive intracellular vesicles is increased when endocytosis is not inhibited by 10°C incubation. n=25 neurons from three cultures for each condition; results are mean±s.e.m. *P≤0.05, **P<0.01 (Student's t-test). Scale bars: 5 μm.

We next investigated the adaptability of γ2pHFAP with other MG dye applications in live-imaging intracellular trafficking assays. The Cy3pH(S/SA)-MG dye is a dichromophore consisting of a pH-sensitive Förster resonance energy transfer (FRET) donor Cy3pH molecule and an acceptor MG (Fig. 5A; Fig. S1) (Perkins et al., 2017 preprint). Excitation of Cy3pH (561 nm excitation) results in highly efficient FRET to MG and emission is observed at 680 nm, while direct stimulation of MG (640 nm excitation) also results in 680 nm emission, allowing calculation of an emission ratio of MG561:MG640 (Fig. 5A). Increasingly acidic environments such as endosomes and lysosomes lead to protonation of Cy3pH and increased fluorescence intensity, enhancing FRET, and the ratio of MG561:MG640 (Grover et al., 2012; Perkins et al., 2017 preprint; Szent-Gyorgyi et al., 2010; Yushchenko et al., 2012). The pH sensor dye is cell impermeant like MG-BTau and thus allows for selective labeling of surface γ2-containing GABAARs. To characterize the pH sensitivity of Cy3pH(S/SA)-MG in our γ2pHFAP neuronal system, we first performed live-cell imaging perfusion experiments using different pH solutions to simulate receptor progression through an increasingly acidic endolysosomal pathway, as similarly performed in other cell culture methods with Cy3pH(S/SA)-MG (Perkins et al., 2017 preprint). Neurons were pulse-labeled with pH sensor dye and then first imaged with pH 7.4 physiological saline, followed by pH 6.8 and pH 4.8 solutions, demonstrating a clear shift in the ratio of MG561:MG640 across treatments (Fig. 5B). Quantification of mean±s.e.m. MG561:MG640 ratios at surface synaptic cluster sites revealed pH 7.4 (0.58±0.01), pH 6.8 (0.69±0.01) and pH 4.8 (0.99±0.02) conditions gave significantly different values from one another (Fig. 5C). These data demonstrate that Cy3pH(S/SA)-MG-labeled γ2pHFAP receptors can be readily identified in environments of different acidities based on their MG561:MG640 ratio.

Fig. 5.

Cy3pH(S/SA)-MG dye bound to surface γ2pHFAP receptors displays a pH-sensitive FRET signal. (A) The pH sensor dye Cy3pH(S/SA)-MG is a fluorogen FRET sensor. Increasingly acidic environment enhances Cy3pH fluorescence intensity and FRET. A higher MG561:MG640 ratio indicates lower pH. (B) DIV 12–13 γ2pHFAP-expressing neurons were pulse-labeled with 100 nM of the pH sensor dye Cy3pH(S/SA)-MG for 2 min, then were immediately used for live imaging. Cells were first perfused with pH 7.4 HBS, then pH 6.8 HBS, and finally MES-buffered saline at pH 4.8. Zoomed dendrite images below highlight MG561:MG640 ratios at surface synaptic clusters across different acidities. All ratiometric images are on a scale from 0.1 to 2. (C) The MG561:MG640 ratio of individual surface γ2pHFAP GABAAR synaptic clusters were quantified at each pH. n=41 synaptic clusters from 4 cells; results are mean±s.e.m. **P<0.01, ***P<0.001, ****P≤0.0001 (one-way ANOVA followed by post hoc Tukey's test). Scale bars: 10 μm (main panels); 2.5 μm (enlargements).

Fig. 5.

Cy3pH(S/SA)-MG dye bound to surface γ2pHFAP receptors displays a pH-sensitive FRET signal. (A) The pH sensor dye Cy3pH(S/SA)-MG is a fluorogen FRET sensor. Increasingly acidic environment enhances Cy3pH fluorescence intensity and FRET. A higher MG561:MG640 ratio indicates lower pH. (B) DIV 12–13 γ2pHFAP-expressing neurons were pulse-labeled with 100 nM of the pH sensor dye Cy3pH(S/SA)-MG for 2 min, then were immediately used for live imaging. Cells were first perfused with pH 7.4 HBS, then pH 6.8 HBS, and finally MES-buffered saline at pH 4.8. Zoomed dendrite images below highlight MG561:MG640 ratios at surface synaptic clusters across different acidities. All ratiometric images are on a scale from 0.1 to 2. (C) The MG561:MG640 ratio of individual surface γ2pHFAP GABAAR synaptic clusters were quantified at each pH. n=41 synaptic clusters from 4 cells; results are mean±s.e.m. **P<0.01, ***P<0.001, ****P≤0.0001 (one-way ANOVA followed by post hoc Tukey's test). Scale bars: 10 μm (main panels); 2.5 μm (enlargements).

Following pH characterization of Cy3pH(S/SA)-MG, we then utilized this dye to identify the localization of internalized γ2pHFAP receptors in the endosomal-lysosomal system. Acidity increases as intracellular receptors undergo transition from early endosomal/recycling pathways to late endosomal/lysosomal pools (Fig. 6A). We therefore anticipated differences in mean MG561:MG640 ratios between these vesicular populations. To examine this effect, we co-transfected γ2pHFAP neurons with the early endosome marker EEA1–GFP for live-imaging experiments. At DIV 12–13, 10 nM Cy3pH(S/SA)-MG was added directly to the cell culture medium and neurons were incubated for 30 min at 37°C prior to imaging. Cells were first perfused with pH 7.4 HBS to acquire MG561:MG640 data, then with pH 6.4 solution to eliminate surface γ2pHFAP (pHGFP) signal (Fig. 6B, orange stars) to isolate intracellular EEA1–GFP for image analysis. Cy3pH(S/SA)-MG vesicles colocalized with EEA1–GFP (Fig. 6B, yellow arrows) demonstrated a mean MG561:MG640 ratio of 0.824±0.012, while all other identified vesicles not-associated with EEA1–GFP (Fig. 6B, white triangles) displayed a significantly higher ratio of 0.918±0.016 (Fig. 6C, other). The Cy3pH(S/SA)-MG found in non-EEA1 intracellular sites typically was present at locations with the lowest pH and was in comparably larger vesicular bodies, suggesting lysosomal compartments. These results indicate Cy3pH(S/SA)-MG-labeled γ2pHFAP receptors can be used to decipher the localization of internalized GABAAR pools along the endosome-lysosome axis.

Fig. 6.

Constitutive endolysosomal trafficking of GABAARs can be measured by using the pH-sensitive dye Cy3pH(S/SA)-MG and γ2pHFAP. (A) Application of pH sensor dye to visualize and distinguish intracellular vesicle targeting of internalized GABAAR. Once internalized, the pHGFP signal is immediately quenched, and the increasingly acidic environment enhances Cy3pH fluorescence intensity and FRET. A higher MG561:MG640 ratio indicates vesicular acidification. Recycling/early endosomal vesicle signal is represented as green and late endosome/lysosomes as red to match the MG561:MG640 ratio heatmap in images. (B) Neurons transfected with γ2pHFAP and EEA1–GFP were incubated in the continuous presence of 10 nM Cy3pH(S/SA)-MG dye for 30 min in conditioned medium at 37°C and then washed and rapidly imaged. After initial images were taken at pH 7.4 and pH 6.4, HBS was perfused onto cells to quench surface pHGFP signal of γ2pHFAP and selectively identify EEA1–GFP-positive vesicles. Yellow arrows indicate vesicles where EEA1–GFP is colocalized with Cy3pH(S/SA)-MG; white triangles indicate larger Cy3pH(S/SA)-MG vesicular structures not colocalized with EEA1–GFP (Other); orange stars represent γ2pHFAP pHGFP surface signal that is eliminated after pH 6.4 saline perfusion. (C) Quantification of individual MG561:MG640 ratios for intracellular vesicles at either EEA1–GFP-positive sites or for other internal compartments (n=243, EEA1–GFP; 511, Other, from 9 cells and 2 neuronal cultures; results are mean±s.e.m.). (D) Confocal images of pH sensor dye-labeled GABAARs at t0, t30 and t30+dynasore (Dyn, 80 μM), with the cell body area measured indicated by the yellow-outlined ROI. Zoomed images of time points are shown to right. (E) More intracellular vesicles are found at t30 than t0 or in the t30+dynasore condition (results are mean±s.e.m.). (F) Histogram analysis reveals most t30 vesicles had lower MG561:MG640 ratios and the median values between all conditions were significantly different (n=14 neurons per treatment from three independent cultures). All ratiometric images are on a scale from 0.1 to 2. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (Student's t-test for C; one-way ANOVA followed by post hoc Tukey's test for E; Mann–Whitney test for F). Scale bars: 10 μm (main panels); 5 μm (enlargements).

Fig. 6.

Constitutive endolysosomal trafficking of GABAARs can be measured by using the pH-sensitive dye Cy3pH(S/SA)-MG and γ2pHFAP. (A) Application of pH sensor dye to visualize and distinguish intracellular vesicle targeting of internalized GABAAR. Once internalized, the pHGFP signal is immediately quenched, and the increasingly acidic environment enhances Cy3pH fluorescence intensity and FRET. A higher MG561:MG640 ratio indicates vesicular acidification. Recycling/early endosomal vesicle signal is represented as green and late endosome/lysosomes as red to match the MG561:MG640 ratio heatmap in images. (B) Neurons transfected with γ2pHFAP and EEA1–GFP were incubated in the continuous presence of 10 nM Cy3pH(S/SA)-MG dye for 30 min in conditioned medium at 37°C and then washed and rapidly imaged. After initial images were taken at pH 7.4 and pH 6.4, HBS was perfused onto cells to quench surface pHGFP signal of γ2pHFAP and selectively identify EEA1–GFP-positive vesicles. Yellow arrows indicate vesicles where EEA1–GFP is colocalized with Cy3pH(S/SA)-MG; white triangles indicate larger Cy3pH(S/SA)-MG vesicular structures not colocalized with EEA1–GFP (Other); orange stars represent γ2pHFAP pHGFP surface signal that is eliminated after pH 6.4 saline perfusion. (C) Quantification of individual MG561:MG640 ratios for intracellular vesicles at either EEA1–GFP-positive sites or for other internal compartments (n=243, EEA1–GFP; 511, Other, from 9 cells and 2 neuronal cultures; results are mean±s.e.m.). (D) Confocal images of pH sensor dye-labeled GABAARs at t0, t30 and t30+dynasore (Dyn, 80 μM), with the cell body area measured indicated by the yellow-outlined ROI. Zoomed images of time points are shown to right. (E) More intracellular vesicles are found at t30 than t0 or in the t30+dynasore condition (results are mean±s.e.m.). (F) Histogram analysis reveals most t30 vesicles had lower MG561:MG640 ratios and the median values between all conditions were significantly different (n=14 neurons per treatment from three independent cultures). All ratiometric images are on a scale from 0.1 to 2. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (Student's t-test for C; one-way ANOVA followed by post hoc Tukey's test for E; Mann–Whitney test for F). Scale bars: 10 μm (main panels); 5 μm (enlargements).

Next, we wanted to elucidate whether Cy3pH(S/SA)-MG could be used to monitor the constitutive endolysosomal trafficking of GABAARs by using conditions that limit internalization. We compared γ2pHFAP neurons pulse-labeled with 100 nM Cy3pH(S/SA)-MG dye either immediately after washing (t0) or 30 min later (t30) with or without the dynamin inhibitor dynasore (80 µM) at 37°C. Importantly, GABAARs have been previously characterized to undergo constitutive dynamin-dependent clathrin-mediated endocytosis (Herring et al., 2003; Kittler et al., 2000a). Images of neurons allowed 30 min of trafficking show enhanced dye-labeled GABAAR accumulation within the cell body compared to t0 and to t30 with dynasore (Fig. 6D). The number of GABAAR-positive vesicles inside the neuronal cell body was measured by using the spot detection feature in NIS Elements. Quantification confirmed that the total number of vesicles identified at t30 is 78.7±20% (mean±s.e.m.) greater than at t0 and that vesicle internalization is blocked by dynasore co-treatment (Fig. 6E). To better understand the receptor population distribution along the endosomal-lysosomal trafficking axis, we examined the respective MG561:MG640 ratio for each vesicle and plotted this data as a histogram (Fig. 6F). This analysis revealed that the increase in vesicles found at t30 were primarily at lower MG561:MG640 ratios, suggesting early endosomal trafficking. Moreover, the vesicle median MG561:MG640 ratios were significantly different between all conditions (t0=1.31; t30=0.767; and t30 with dynasore=0.826). Interestingly, we found that although dynasore reduced the number of internalized receptors detected back to t0 levels, dynasore-treated neurons demonstrated a unique vesicular MG561:MG640 ratio profile (Fig. 6E). Dynasore inhibition of clathrin-coated pit formation and endocytosis generates half formed ‘U’ and ‘O’ shaped pits/vesicles associated with the plasma membrane (Macia et al., 2006). The few vesicles present in t30 with dynasore neurons (Fig. 6D) are likely to be these structures undergoing partial acidification during the 30 min experimental period. In summary, γ2pHFAP and the Cy3pH(S/SA)-MG dye reveal significant constitutive clathrin-dependent endocytosis of GABAARs that favors early endosomal pathways.

γ2pHFAP reveals enhanced GABAAR synaptic turnover in a bicuculline seizure paradigm

We last set out to investigate whether the γ2pHFAP–dye system could be used to measure pharmacologically induced changes in GABAAR trafficking in living neurons. Prolonged exposure of the GABAAR antagonist bicuculline is proconvulsant due to sustained dampening of network inhibition, leading to seizure-type activity in vitro and in vivo (Chauvette et al., 2016; Colombi et al., 2013; Hongo et al., 2015; Khalilov et al., 1997). Multiple hyperexcitable neuronal states have previously been reported to enhance γ2-containing GABAAR internalization and reduce total surface levels at 1 h post induction (Goodkin et al., 2008, 2005; Naylor et al., 2005; Terunuma et al., 2008). We investigated whether the γ2pHFAP construct could be used to simultaneously examine multiple stages of receptor trafficking including receptor surface, synaptic and lysosomal levels following a bicuculline-induced seizure paradigm. At DIV 12–14 γ2pHFAP neurons were pulse-labeled with MG-BTau dye and then returned to conditioned medium with or without 50 µM bicuculline at 37°C for 1 h. 50 nM LysoTracker was added 30 min prior to the end of treatment to identify association of receptors with lysosomes. Representative images indicate that MG-BTau labels synaptic GABAAR clusters on the surface of dendrites as seen by colocalization of MG-BTau (blue) and pHGFP (green) (Fig. 7A,B). MG-BTau also reveals internalized receptors within the cell body in lysosomes (Fig. 7C, Lysotracker in red). These data demonstrate that the binding of MG-BTau to γ2pHFAP GABAARs and its resulting fluorescence is stable even in very low pH environments, such as lysosomes, consistent with previous findings using different FAP-tagged receptors colocalized with LysoTracker in cell culture (Grover et al., 2012). Moreover, we detect sustained MG-BTau signal and labeling of surface γ2pHFAP during extracellular pH 7.4 to 4.8 solution exchange experiments (Fig. S2). Image analysis uncovered no significant difference in total surface expression of γ2pHFAP between DMSO control and bicuculline-treated cells when measuring the pHGFP signal (Fig. 7D). There was a trend towards a decrease in synaptic levels of γ2pHFAP in bicuculline-treated neurons (75±13% of control; mean±s.e.m.), as determined by pHGFP cluster fluorescence, but this was not significant. In contrast, Fig. 7E shows that bicuculline treatment reduced total and synaptic MG-BTau signal by 41±10% and 67±8%, respectively, indicating the population of pulse-labeled γ2pHFAP receptors had decreased. In support of enhanced receptor turnover, we found that bicuculline treatment increased association of labeled receptors with lysosomes by 107±41% compared to control. These findings suggest that bicuculline-induced seizure activity leads to augmented GABAAR synaptic turnover, lysosomal targeting, and a compensatory increase in new (non-recycled) GABAAR insertion to mitigate this response. These results robustly demonstrate the versatility of the γ2pHFAP–dye system and its ability to measure numerous trafficking events to address complex biological questions.

Fig. 7.

γ2pHFAP imaging reveals increased internalization and enhanced GABAAR turnover rates following a bicuculline-induced seizure paradigm. (A) γ2pHFAP neurons were pulse-labeled with 100 nM MG-BTau for 2 min then returned to 37°C conditioned medium with or without 50 µM bicuculline for 1 h. LysoTracker (50 nM) was added directly to the medium after 30 min to label lysosomes. pHGFP fluorescence is shown in green, LysoTracker in red and MG-BTau in blue in the Merge panels. Smaller boxed areas in the Merge panel identify surface synaptic receptors (enlarged in B). Larger boxed area identifies internalized receptors present in endosomes and lysosomes in cell body of neuron (enlarged in C). (B) Surface synaptic receptors on dendrites are seen with colocalization of MG-BTau and pHGFP signals. (C) Enlargements of cell body area show colocalization of internalized MG-BTau labeled GABAARs and lysosomes (yellow arrowheads). (D) Quantification of the pHGFP signal showing that synaptic and total surface levels were not changed following bicuculline treatment. (E) In contrast, quantification of MG-BTau signal revealed reductions in total and synaptic receptor levels after the bicuculline seizure paradigm. Bicuculline treatment also enhanced the proportion of MG-BTa-labeled receptors associated with lysosomes more than the total MG-BTau signal. n=13 neurons per treatment from three independent cultures; results are mean±s.e.m. Synaptic measurements were performed on three 10 μm regions located on dendrites. *P<0.05, ****P<0.0001 (Student's t-test). Scale bars: 20 μm (A); 2 μm (B,C).

Fig. 7.

γ2pHFAP imaging reveals increased internalization and enhanced GABAAR turnover rates following a bicuculline-induced seizure paradigm. (A) γ2pHFAP neurons were pulse-labeled with 100 nM MG-BTau for 2 min then returned to 37°C conditioned medium with or without 50 µM bicuculline for 1 h. LysoTracker (50 nM) was added directly to the medium after 30 min to label lysosomes. pHGFP fluorescence is shown in green, LysoTracker in red and MG-BTau in blue in the Merge panels. Smaller boxed areas in the Merge panel identify surface synaptic receptors (enlarged in B). Larger boxed area identifies internalized receptors present in endosomes and lysosomes in cell body of neuron (enlarged in C). (B) Surface synaptic receptors on dendrites are seen with colocalization of MG-BTau and pHGFP signals. (C) Enlargements of cell body area show colocalization of internalized MG-BTau labeled GABAARs and lysosomes (yellow arrowheads). (D) Quantification of the pHGFP signal showing that synaptic and total surface levels were not changed following bicuculline treatment. (E) In contrast, quantification of MG-BTau signal revealed reductions in total and synaptic receptor levels after the bicuculline seizure paradigm. Bicuculline treatment also enhanced the proportion of MG-BTa-labeled receptors associated with lysosomes more than the total MG-BTau signal. n=13 neurons per treatment from three independent cultures; results are mean±s.e.m. Synaptic measurements were performed on three 10 μm regions located on dendrites. *P<0.05, ****P<0.0001 (Student's t-test). Scale bars: 20 μm (A); 2 μm (B,C).

DISCUSSION

Live-cell receptor tracking approaches offer critical information by revealing real-time alterations in protein trafficking. Here, we describe a flexible paired GABAAR γ2 subunit optical reporter system that can be used to monitor multi-stage receptor trafficking. Compared to previously available methods that require reliable antibodies, conventional fluorophore tagging or fluorescent α-bungarotoxin labeling, the γ2pHFAP–dye system can allow for simultaneous monitoring of surface, synaptic and intracellularly trafficking GABAARs in real-time. The strength of this method lies in the ability to simply switch FAP-compatible MG dyes to address the specific experimental question of interest.

We applied the pH sensor dye Cy3pH(S/SA)-MG to monitor internalization and constitutive endosomal and lysosomal trafficking of GABAARs in neurons. We found that Cy3pH(S/SA)-MG-labeled surface γ2pHFAP receptors can be tracked to small EEA1-positive early endosomes (MG-BTau-labeled receptors were also identified at EEA1 endosomes; Fig. 4) and also to larger vesicular structures with low pH (likely lysosomes), generating high MG561:MG640 ratios (Fig. 6B,C). Synaptic GABAAR endocytosis occurs primarily in a dynamin-dependent manner (Herring et al., 2003) and can be identified within 30 min when using an antibody-feeding approach (Kittler et al., 2000a). Our results in Fig. 6 demonstrate rapid internalization of receptors in endosomal/lysosomal pathways on this same time scale, a process reduced by the dynamin-inhibitor dynasore. As a result of the vesicle-level pH-sensitive measurement afforded by this tool, we were able to further generate a histogram plot identifying differences in the trafficking stages of γ2pHFAP-positive vesicles. These analyses suggest that internalized receptors favor early endosomal pathways, a finding supported by a previous study that determined that over 70% of internalized GABAARs are recycled back to the cell surface within 1 h (Kittler et al., 2004). Furthermore, dynasore-treated neurons displayed vesicles with a distinct MG ratio profile, possibly resulting from acidification of endocytic pits/vesicles not released from the plasma membrane, non-dynamin-dependent endocytosis of receptors or off-target effects of dynasore. GABAARs undergo both clathrin-dependent and -independent endocytosis pathways in heterologous cells (Cinar and Barnes, 2001), with both mechanisms being recently identified in neurons for glutamateric α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Glebov et al., 2015; Zheng et al., 2015). Dynasore has also been shown to disrupt lysosomal fission by inhibiting dynamin-2 (Schulze et al., 2013) as well as having other non-specific dynamin-independent effects (Park et al., 2013; Preta et al., 2015) that could result in different vesicle MG561:MG640 ratio profiles. Further studies investigating additional time points are necessary to identify the rate, equilibrium and overall population dynamics of constitutive GABAAR internalization.

To test the utility of γ2pHFAP in detecting pharmacologically induced changes in GABAAR trafficking, we exposed neurons to a bicuculline-induced seizure paradigm following a MG-BTau pulse-labeling protocol (Fig. 7). This unique dye labeling approach readily detected enhanced synaptic receptor turnover rates and lysosomal targeting that were not detectable by pHGFP fluorescence alone. Previous work has shown that more aggressive seizure protocols relying on depolarization via high external K+ and/or agonist application targeting highly Ca2+-permeable N-methyl-D-aspartic acid (NMDA) receptors reduces total surface GABAARs in cultured neurons (Goodkin et al., 2008). It is likely that the mild seizure paradigm used here does not affect total surface receptor levels, but instead enhances surface turnover that is offset by increased receptor insertion rates. In support of this argument, another recent study (Chaumont et al., 2013) found that a 1 h treatment with GABAAR antagonists did not lead to a reduction in surface GABAAR levels.

This paper lays the groundwork for future investigations of GABAAR trafficking with the γ2pHFAP sensor. Importantly, this technique can be further extended toward pharmacology-focused efforts in high-throughput screenings (Fisher et al., 2014; Snyder et al., 2015), assay development based on flow cytometry (Saunders et al., 2012; Wu et al., 2014) or high-resolution 96-well plate assays (Larsen et al., 2016) and for in vivo protein labeling (Liu et al., 2016; Zhang et al., 2015). In summary, our γ2pHFAP is the first protein–FAP conjugate characterized in primary neurons, providing a unique tool to monitor multistage GABAAR trafficking in living cells with relevance both for basic science research and applied pharmacology.

MATERIALS AND METHODS

Cell culture and transfection

All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Cortical neurons were prepared from embryonic day 18 rats (Sprague Dawley rat, Charles River) and nucleofected (Lonza, Switzerland) at plating (Jacob et al., 2005). HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) and were transfected by nucleofection.

DNA constructs and antibodies

The α2, β3 and pH-sensitive GFP-tagged γ2 subunit (γ2pHGFP) plasmids have been previously described (Bedford et al., 2001; Jacob et al., 2005; Kittler et al., 2000b; Tretter et al., 2008). The flurogen-activating peptide dl5 (Szent-Gyorgyi et al., 2013) was inserted upstream of pHGFP (γ2pHFAP) separated by a G-A-P-P-A amino acid linker. The EEA1–GFP construct was Addgene plasmid #42307 (deposited by Silvia Corvera) (Lawe et al., 2000). All constructs were sequenced to confirm the fidelity of final plasmids. The following primary antibodies were used: mouse anti-β-actin (1:2000, A1978, Sigma); rabbit anti-GFP (1:1000, A11122, Invitrogen); rabbit anti-VGAT (1:1000, 131002, Synaptic Systems); mouse anti-EEA1 (1:1000, 610457, BD Biosciences) antibodies. Immunofluorescence secondary antibodies were: goat anti-rabbit-IgG conjugated to Alexa Fluor 641 (1:1000, A21245, Invitrogen); goat anti-mouse-IgG conjugated to Alexa Fluor 405 (1:1000; A31553, Invitrogen).

MG dyes

MG dyes were kindly provided by Dr Alan S. Waggoner, Dr Ming Zhang, Dr Marcel P. Bruchez, and Dr Brigitte F. Schmidt at Carnegie Mellon University, Pittsburgh, PA. The MG dye MG-BTau was synthesized as described previously (Grover et al., 2012; Yan et al., 2015) and the MG dye Cy3pH(S/SA)-MG was prepared by the method of Perkins et al. (2017 preprint). The IUPAC name for Cy3pH(S/SA)-MG is 2-((E)-3-((Z)-3,3-dimethyl-5-sulfoindolin-2-ylidene)prop-1-en-1-yl)-1-(6-((3-(4-((4-(dimethylamino) phenyl)(4-(dimethyliminio)cyclohexa-2,5-dien-1-ylidene)methyl)phenoxy)propyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfamoyl-3H-indol-1-ium. Structural, synthetic and analytical details for MG-BTau are as described previously (Larsen et al., 2016; Pratt et al., 2015; Yan et al., 2015).

Immunocytochemistry and confocal microscopy

Primary cortical neurons grown on glass coverslips were fixed at days in vitro (DIV) 13–14. Neurons were permeabilized and stained with anti-VGAT antibody. Images were taken on a Nikon Ti-E A1 confocal microscope equipped with a motorized Z-stage and perfect focus system (PFS) using a 60× oil immersion objective (NA 1.49) at 3× zoom. Data were analyzed by using NIS Elements software (Nikon, NY). Thresholds were set using binary masks to selectively identify brightly fluorescent objects above background (Jacob et al., 2005). Three dendritic 10 µm regions of interest (ROI) were drawn per neuron to measure synaptic colocalization of pHGFP signal with VGAT in γ2pHFAP characterization studies. γ2pHGFP control and γ2pHFAP-expressing neuron pixel intensity and sum area of synapses in µm2 was measured and values were normalized to control mean. Fixed EEA1 early endosome studies utilized a 2-min 100 nM MG-BTau dye pulse-labeling protocol in HBS at room temperature. After live-cell MG dye incubation steps, cells were washed five times to remove all unbound dye prior to returning the cells to 10°C or 37°C solution for 30 min followed by fixation and immunostaining. The total pixel intensity of MG-BTau-labeled receptors colocalized with EEA1 was measured within a cell body ROI. Laser settings were held constant across experiments. Image acquisition and data analysis was carried out by a researcher who was blind to the experimental details.

Live-cell imaging

Transfected DIV 12–14 cortical neurons or HEK293 cells were plated on MatTek glass-bottom dishes (Ashland, MA). Imaging was performed in HEPES-buffered saline (HBS): 135 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 10 mM HEPES, 2.5 mM CaCl2, 11 mM glucose, pH 7.4 (Jacob et al., 2012). Low-pH HBS (pH 6.4–6.8) was similarly prepared, while pH 4.8 saline was a MES-buffered saline: (135 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 10 mM MES, 2.5 mM CaCl2, 11 mM glucose). All dye pulse-labeling steps were performed in HBS at room temperature. Following dye incubation steps, cells were washed five times to remove all unbound dye prior to treatment or imaging. All images were taken at room temperature using a 60× objective at a 3× zoom within 10 min of dye washout. Perfusion assays utilizing different pH saline buffers monitored a single cell per assay, while two or three cells were imaged in each experimental dish for all other experiments. Data analysis was carried out by a researcher who was blind to the experimental details where possible.

Cy3pH(S/SA)-MG dye was used for assays of constitutive trafficking in neurons. Neurons were pre-incubated in DMSO vehicle control (t0, t30) or 80 µM dynasore (2897, Tocris) 30 min prior to pulse-labeling. After dye exposure, neurons were either immediately imaged (t0) or returned to conditioned medium with or without the continued presence of dynasore for 30 min at 37°C prior to imaging. Cy3pH(S/SA)-MG dye acquisition measurements were taken sequentially to measure the Cy3pH-induced (561 nm excitation) MG emission (680 nm) followed by MG-induced (640 nm excitation) MG emission (680 nm). Laser settings were then switched to allow capture of pHGFP signals (488 nm excitation; 510 nm emission). Individual vesicles were identified using the NIS Elements Spot Detection tool and thresholding analysis. A ROI was drawn around the cell body and binary thresholds were set to selectively capture only pHGFP clusters and the MGex640 (MG signal excited by 640 nM) signal above background. Next, spot detection thresholds were set to selectively identify the MGex561 (signal excited by 561 nM) fluorescent objects with a minimum circular area of 0.55 µm2 and signal above threshold. Colocalization of the MGex561 spot signal and MGex640 binary signal was considered as a γ2pHFAP-positive vesicle. To remove contributions of surface synaptic GABAARs from these vesicle measurements, MGex561 and MGex640 signals colocalized with pHGFP signals were subtracted. The values reported reflect the total number of MGex561- and MGex640-positive objects (vesicles) identified and the MG561:MG640 ratio of each vesicle. Early endosome characterization assays measured colocalization of Cy3-MG objects with EEA1–GFP vesicles to determine the MG561:MG640 ratio in these compartments, while all non-associated Cy3-MG objects were placed in the ‘other’ category. A ROUT outlier test (Q=1.0%) was used in this experimental analysis. For surface Cy3pH(S/SA)-MG pH characterization perfusion assays, the ratio of MG561:MG640 was determined by selectively measuring synaptic clusters of γ2pHFAP GABAARs.

Neuron surface and lysosomal-association assays utilized MG-BTau dye for surface receptor pulse-labeling. For bicuculline-induced seizure assays, neurons were either immediately imaged following 100 nM MG dye incubation (t0) or returned to conditioned medium in the presence of 50 µM (–)-bicuculline methiodide (2503, Tocris) or DMSO vehicle control. To label lysosomes, neurons were incubated at 37°C in 50 nM LysoTracker Blue DND-22 (Life Technologies) in conditioned medium 30 min prior to imaging. For image analysis, independent ROIs were drawn to capture the soma, three 10 µM sections of dendrite and the whole cell. Binary thresholds and colocalization measurements were performed as above to identify MG-BTau, pHGFP synaptic GABAAR clusters and lysosomes. Total surface pHGFP expression was determined by taking the entire cell surface signal following background subtraction.

Western blotting

Transfected and non-transfected DIV 13-14 cortical neurons were lysed with RIPA buffer containing: 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 2 mM sodium orthovanadate and protease inhibitor cocktail (Sigma, St. Louis, MO), solubilized for 15–30 min at 4°C, and spun at 13,500 g for 15 min to remove the nuclear pellet. A BCA protein assay was performed on the supernatant, and equivalent amounts of protein were loaded for SDS-PAGE analysis. After electrophoresis and transfer to nitrocellulose membrane, samples were probed with primary antibody overnight followed by the appropriate horseradish peroxide (HRP)-coupled secondary antibody. Blots were visualized by using a Biorad Chemicdoc XRS+ following ECL development (Thermo Scientific).

Electrophysiology

Whole-cell voltage-clamp recordings were performed on HEK293 cells 12–48 h after nucleofection with GABAAR subunits. Cells were transfected with (1) EGFP:α2:β3:γ2pHGFP or (2) EGFP:α2:β3:γ2pHFAP in a 1:1:1:3 ratio to favor the production of γ2 subunit-containing receptors. Pipettes were pulled from borosilicate capillary tubing (Sutter Instruments) to a resistance of 2–5 MΩ on a Sutter Instruments-Flaming Brown P-97 electrode puller and fire polished. Unless otherwise indicated, the extracellular solution contained (in mM): 140 NaCl, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 10 HEPES and 11 D-glucose, and was pH adjusted to 7.4±0.05 with NaOH. The intracellular solution contained (in mM): 140 CsCl, 0.1 CaCl2, 10 HEPES, 1.1 EGTA, 2 MgCl2, 2.5 phosphocreatine, 2 ATP-Mg and 1 GTP-Na, and was pH adjusted to 7.2±0.05 with CsOH. Recordings were made from cells expressing EGFP as identified by epifluorescence illumination on an inverted Zeiss Axioscope microscope. Cells were held at a membrane potential (Vm) of −50 mV for all experiments. Vm was corrected for an empirically determined liquid junction potential between the extracellular and intracellular solution of −4 mV. Whole-cell currents were recorded using an Axopatch 200A patch-clamp amplifier (Molecular Devices), low-pass filtered at 5 kHz and sampled at 10 kHz in pClamp10.7 (Molecular Devices). Series resistance was compensated for with the prediction and correction circuitry to at least 75% in all experiments. Rapid solution exchange was achieved using an in-house-fabricated 10-barrel fast perfusion system connected to gravity-fed reservoirs similar to the system previously described (Blanpied et al., 1997). All experiments were performed at room temperature. Peak currents were measured relative to baseline current prior to agonist application using Clampfit 10.7.

Statistics

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA) or Microsoft Excel. Unpaired Student's t-tests or one way ANOVA with subsequent post hoc Tukey's test were used to determine significance in indicated imaging studies. Significant differences in data distribution for Cy3pH(S/SA)-MG experiments were determined using the Mann–Whitney test. Paired or unpaired two-tailed Student's t-tests were used to determine significance of differences in electrophysiological studies. All data are reported as mean±s.e.m. unless otherwise indicated in the text.

Acknowledgements

We would like to thank Megan Brady for her technical assistance during the design and generation of the γ2pHFAP construct.

Footnotes

Author contributions

Conceptualization: J.M.L.-G., M.R.W., M.Z., J.W.J., A.S.W., T.C.J.; Methodology: J.M.L.-G., M.R.W., M.Z., M.B.L., J.W.J., A.S.W., S.C.W.; Validation: J.M.L.-G., M.R.W., M.B.L., J.P., T.C.J.; Formal analysis: J.M.L.-G., M.R.W., J.W.J.; Investigation: J.M.L.-G., M.R.W., J.P.; Resources: M.Z., M.B.L., B.F.S., M.P.B., J.W.J., A.S.W., S.C.W., T.C.J.; Data curation: J.M.L.-G., M.R.W., J.W.J., T.C.J.; Writing - original draft: J.M.L.-G.; Writing - review & editing: J.M.L.-G., M.R.W., M.B.L., M.P.B., J.W.J., A.S.W., S.C.W., T.C.J.; Visualization: M.R.W., T.C.J.; Supervision: J.W.J., T.C.J.; Project administration: J.M.L.-G., T.C.J.; Funding acquisition: T.C.J.

Funding

This work was generously funded by a National Institute of Health Training Grant (T32GM008424), the National Institute of Mental Health (R01 MH045817), a National Alliance for Research on Schizophrenia and Depression young investigator grant, a University of Pittsburgh Pharmacology and Chemical Biology Fellowship (William C. deGroat Neuropharmacology Departmental Fellowship) and Pharmacology and Chemical Biology Startup Funds. Deposited in PMC for release after 12 months.

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Competing interests

M.P.B. is a founder and Chief Scientific Officer of Sharp Edge Labs, Inc., a company that is using the FAP–fluorogen technology commercially. A.S.W. is an advisor for Sharpe Edge Labs with financial involvement. Sharp Edge Labs is producing assays based on fluorogen-activating peptides, but is not investigating GABAAR-related research or drug development.

Supplementary information