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. Author manuscript; available in PMC: 2026 May 6.
Published in final edited form as: J Vis Exp. 2025 Dec 19;(226):10.3791/69371. doi: 10.3791/69371

Localization of Plasma Membrane and Intracellular Neuronal Nicotinic Acetylcholine Receptors Using Quantitative Imaging in Mammalian Cells

Lahra Weber 1, Sarah M Suarez 1, Maegan M Weltzin 1,2
PMCID: PMC13143191  NIHMSID: NIHMS2166663  PMID: 41490023

Abstract

Quantification of neuronal nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane of mammalian cells is important for the development of novel target-selective therapeutics and for studying chaperone proteins that modulate receptor trafficking. This study presents a robust set of methodologies to express both homomeric and heteromeric nAChRs in mammalian Neuro2a (N2a) cells and to quantify their surface versus intracellular localization using pH-sensitive fluorescent tags. pHuji and superecliptic pHluorin (SEP) are pH-sensitive proteins that can be engineered to tag recombinant proteins, allowing visualization of the spatial distribution of nAChR subunits. These fluorescent tags emit at neutral pH but are quenched in acidic environments. To track the relative internal versus plasma membrane localization of α7, α4, and β2 nAChR subunits, either pHuji or SEP is attached to the C-terminus of each DNA construct. To express α7-pHuji together with its chaperone NACHO, or (α4-SEP; β2-pHuji) in N2a cells, a lipid-based DNA transfection reagent is used. The transfection reagent and plasmid DNA are incubated separately in reduced-serum medium at room temperature, then combined to form lipid-DNA complexes that facilitate plasmid delivery into cells. Following incubation, cells are imaged using live-cell confocal microscopy under both high- and low-pH conditions or in the presence of a subtype-selective fluorescent ligand. Images are analyzed using corrected total cell fluorescence in the relevant fluorophore channels, providing quantitative information on the distribution of fluorescently tagged nAChR subunits between the plasma membrane and intracellular compartments. Live-cell confocal imaging enables real-time tracking of nAChR subunit localization to distinguish differences between internal and surface receptor populations. Robust plasma membrane expression of α7-pHuji or (α4-SEP; β2-pHuji) is achieved within 24 h at 37 °C. This method can be adapted to include additional chaperones, auxiliary subunits, or fluorescent ligands for the study of nAChR trafficking, pharmacological modulation, and disease-related alterations in nAChR expression.

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated transmembrane ion channels expressed on the plasma membrane of many cell types, including neurons, glial cells, and immune cells. These receptors are composed of α (α2-α10) or β (β2-β4) subunits that assemble to form homomeric or heteromeric subtypes, with α7 and α4β2 nAChRs being the most abundant within the brain1,2. Dysregulation of these subtypes has been associated with Alzheimer’s disease, nicotine use disorder, sleep-related hypermotor epilepsy, Parkinson’s disease, and major depressive disorder, among other neurological and psychiatric conditions3,4,5,6,7,8,9,10,11,12. Rapid and reliable methods to express and quantify nAChRs are essential for understanding disease mechanisms and for developing target-selective therapeutics. However, achieving robust plasma membrane expression of these receptors is often challenging and time-consuming. The method described here enables strong surface expression of nAChRs within approximately 24 h after DNA transfection and can be combined with ligands or chaperone proteins that modulate receptor trafficking.

Several approaches exist for expressing nAChRs, including transient DNA transfection or the generation of stable cell lines that express the desired subtype. Although creating stable cell lines is useful for long-term studies involving the same receptor subtype, the process is labor-intensive and can require several months. Once established, such cell lines may exhibit variable receptor expression across passages, loss of expression over time, or selection bias for clones with suboptimal receptor function or density13. A reduction in incubation temperature from 37 °C to 30 °C, the addition of molecular chaperones such as nicotine, or extended incubation time can promote surface expression13,14,15. These limitations are particularly pronounced for heteromeric nAChRs, which require proper subunit assembly and trafficking, and for studies focusing on chaperone-assisted receptor regulation. Therefore, routine validation of receptor subtype expression across passages is necessary when working with stable nAChR-expressing cell lines.

Often, researchers are interested in nAChR subunit variants and chaperone or auxiliary protein subunits and thus need to express a variety of DNAs in quick succession to allow for rapid flexibility in their assays. Transient expression of nAChRs is advantageous in this regard because the process can be completed rapidly, allowing the researcher to study multiple receptor subtypes or experimental conditions without waiting months to generate stable cell lines. A common occurrence, even with transient expression, is that many nAChRs remain inside the cell 24 h after induction at 37 °C, requiring additional incubation periods of > 48 h16,17. For example, without the chaperone protein resistant to inhibitors of cholinesterase (RIC)-3, only ~1% of α7 nAChRs are expressed on the plasma membrane of mammalian SHEP cells18. With co-expression of RIC-3, approximately 20% of α7 nAChRs are detected on the surface. While this represents an improvement, total receptor abundance remains low. Co-expression of α7 nAChRs with NACHO can further enhance surface expression, though still less than 40% of the total receptor population traffics to the plasma membrane19. Another approach involves incubating transfected cells at 30 °C to improve plasma membrane expression20,21. For α4β2 nAChRs, this temperature reduction yields a fivefold upregulation in surface receptor density with no corresponding increase in total subunit protein21. However, this improvement requires at least one additional day and greater use of consumables, reagents, and instrumentation.

The presented work offers a set of optimized methods to express and quantify specific nAChR subtypes within 24 h post-transfection in mammalian N2a cells, achieving approximately 83% for α7, 77% for α4, and 56% for β2 subunits localized to the plasma membrane. In the case of α7 nAChRs, plasma membrane expression can be quantified using α7-selective fluorescently labeled α-bungarotoxin (αBTX) or through recombinant fluorescent tagging. To label each subunit, the pH-sensitive fluorescent proteins pHuji and superecliptic pHluorin (SEP)22,23,24 are engineered onto the C-terminus of individual nAChR subunits25,26. pHuji and SEP are pH-sensitive variants of red and green fluorescent proteins, respectively, that fluoresce at pH 7.4 but are quenched under more acidic conditions27,28. As SEP and pHuji fluorescence is suppressed at pH < 6, any observed intracellular signal reflects fluorophores located in neutral compartments rather than on the plasma membrane. It should be noted that some organelles, such as the Golgi apparatus, have an acidic lumen; thus, fluorophores attached to the C-terminal region of nAChR subunits are not detected while subunits transit through the secretory pathway29. The difference between total fluorescence at pH 7.5 and that at pH < 6 represents the signal generated by nAChRs located on the plasma membrane. Capturing images using live-cell confocal microscopy provides high spatial resolution, enabling precise quantification of subunit localization. Additionally, performing all experiments on live cells allows each cell to serve as its own control, accounting for variability in protein expression between cells within each treatment group30. This method can be further adapted to include additional chaperone proteins, such as NACHO or others, to investigate nAChR trafficking. Additional applications include pharmacological modulation, incorporation of auxiliary subunits, and analysis of disease-associated changes in nAChR expression.

Protocol

NOTE: The following procedure describes methods that have been used successfully to transiently transfect the N2a cell line with nAChR subunits (Figure 1). Robust plasma membrane expression is achieved within 24 h, as captured with live-cell confocal microscopy.

Figure 1: Schematic presentation of the individual methods presented in the protocol.

Figure 1:

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Cells are grown until they are 70%−80% confluent in imaging dishes. The cells are then treated with the transfection reagent and the DNA complex. (Top) 24 h later, cells can be treated with a fluorescently tagged ligand to label subunits expressed on the plasma membrane and then imaged using a confocal microscope. (Bottom) Alternatively, for nAChR subunit genes that have been modified to be tagged with a pH-sensitive protein 24 h after transfection, cells can be imaged alive using a confocal microscope to capture the total fluorescence of the cells. The pH 7.4 imaging buffer is then changed to the pH 5.5 quenching buffer. This will stop the fluorescence of the pH-sensitive proteins tagged to the nAChR subunits located on the plasma membrane of the cell, allowing for only the internal subunits to remain fluorescent. The same cells are again imaged. Image analysis is performed using ImageJ to quantify the expression of internal and external subunits. These procedures can be modified to incorporate chaperone and auxiliary proteins. Created in BioRender.

1. N2a nAChR transient transfection

NOTE: The N2a cell line was chosen because it exhibits a neuronal phenotype and low endogenous expression of nAChRs. The α7-pHuji, α4-SEP, and β2-pHuji subunit genes are each housed individually in the mammalian pcDNA3.1(+) expression vector. The chaperone protein NACHO is expressed from pREP9 (generously gifted by Dr. R. Loring, Northeastern University). In all steps, use sterile techniques and work inside a biosafety cabinet. Wear a clean laboratory coat, safety glasses, and gloves. Clean all materials entering or leaving the biosafety cabinet with 70% ethanol. The following steps describe transfection in a 35 mm poly-D-lysine-coated glass-bottom dish, which is suitable for imaging. Volumes may be adjusted as needed for different culture vessels.

  1. Culture N2a cells in a T25 (or similar) tissue culture flask containing Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and, if required, 100 U/mL penicillin and 100 μg/mL streptomycin. Incubate at 37 °C, 5% CO2 until cells reach ~90% confluency. Visualize using a 4x light-microscope objective.

  2. Monitor confluency daily using a light microscope and replace the growth medium every 1–2 days to maintain optimal conditions. When cells reach ~90% confluency, they are ready to be passaged into imaging dishes.

  3. Prewarm all reagents to 37 °C in a water bath. Clean the biosafety cabinet and all materials with 70% ethanol before beginning the procedure.

    CAUTION: 70% ethanol is a highly flammable liquid and vapor that can cause serious eye irritation. Keep away from heat, sparks, open flames, and other ignition sources. Store in a well-ventilated area with containers tightly closed. Wear protective gloves and eye/face protection when handling. In case of contact with skin or eyes, rinse immediately with water and seek medical attention if irritation persists. An alternative disinfectant, such as a 5%−10% bleach solution, may be used instead.

  4. Aspirate the old medium from the flask and wash cells with 5 mL of PBS. Add 1 mL of trypsin to the flask and gently swirl. Incubate until cells lift (<5 min). To prevent over-digestion, immediately add 4 mL of EMEM growth medium and gently pipette to create a single-cell suspension.

  5. Using a hemocytometer, determine the cell concentration and calculate the required volume of EMEM growth medium to adjust the suspension so that 1 mL contains 0.5–2 × 105 cells.

  6. Plate 1 mL of cell suspension per 35 mm dish and add enough EMEM growth medium to bring the total volume to 2 mL. Incubate overnight at 37 °C, 5% CO2. Cells are ready for transfection at 70%−80% confluency.

  7. On the day of transfection, prewarm reagents to 37 °C, thaw nAChR subunit DNAs completely, and bring the transfection reagent to room temperature. Clean all reagents with 70% ethanol before placing them in the biosafety cabinet.

  8. To express α7 nAChRs: In a microcentrifuge tube, dilute 4 μg of α7 (1 μg/μL) and 1 μg of NACHO (1 μg/μL) plasmid DNAs dropwise into reduced serum medium (no antibiotic) to a final volume of 250 μL. Mix gently by pipetting after DNA addition.

    1. To express α4β2 nAChRs: Dilute 4 μg of each subunit DNA in a total of 250 μL of reduced serum medium. Mix gently by pipetting. Adjust the ratio of each subunit to optimize expression of specific receptor isoforms.

    2. To express α4β2 nAChRs with an additional plasmid, add equal amounts of each plasmid DNA. The blue fluorescent protein mTagBFP231 in pcDNA3.1(+) serves as a control plasmid and can be replaced with any α4β2 chaperone.

  9. Gently mix the DNA transfection reagent before use. In a separate microcentrifuge tube, dilute 8 μL of transfection reagent dropwise in 250 μL of reduced serum medium. Incubate for 5 min at room temperature. Proceed to the next step within 25 min.

  10. After 5 min, combine the diluted DNAs with the diluted transfection reagent (total = 500 μL) dropwise. Mix gently by pipetting and incubate for 20 min at room temperature. DNA-lipid complexes remain stable for up to 6 h.

  11. While the complexes form, remove culture dishes from the incubator and replace the medium with 1 mL of serum-free EMEM (no antibiotics).

  12. Add 500 μL of the DNA-transfection reagent complex dropwise to each dish (final volume = 1.5 mL). Mix gently by rocking the plate to ensure even distribution across the dish, particularly over the glass-bottom surface. Incubate at 37 °C for 24 h, then replace the medium after 4–6 h to promote cell health.

2. Live cell labeling of plasma membrane-expressed α7 nAChRs using αBTX Alexa Fluor 647

NOTE: The following procedure is generally performed 24 h after transfection. Protect αBTX Alexa Fluor 647 (αBTX-AF647) from light to prevent photodestruction of the fluorophore.

  1. Prewarm all reagents to 37 °C and thaw the αBTX-AF647 stock (2.3 μM) solution. Clean with 70% ethanol and place in the biosafety cabinet.

  2. Retrieve the dish containing the cells transfected with α7 and NACHO DNAs from the incubator, clean the outside with 70% ethanol, and place it in the biosafety cabinet.

  3. Discard the old medium by vacuum suction. To wash the cells, add 2 mL of fresh EMEM growth medium.

  4. Remove the wash medium by vacuum suction. Add 2 mL of EMEM growth medium to the dishes.

  5. Replace the appropriate amount of medium with αBTX-AF647 to achieve a final concentration of 80 nM. Incubate overnight at 37 °C.

  6. In the morning, prewarm PBS or a preferred imaging medium and clean with 70% ethanol. Transfer to the biosafety cabinet.

  7. Remove transfected cells from the incubator, clean, and place them in the biosafety cabinet.

  8. Remove the old medium containing αBTX-AF647 and place it in an appropriate waste container.

  9. Wash cells with 2 mL of PBS for 3x, 10 min each at room temperature. The rinses remove unbound ligand and debris, and the time can be modified to meet the researcher’s needs. Remove PBS after each wash.

  10. Add 2 mL of PBS or a preferred imaging medium and proceed to image cells using a confocal microscope.

3. Live-cell imaging of total and internal α7-pHuji nAChRs

NOTE: The following procedure uses an in-house-made α7-pHuji pcDNA3.1 plasmid co-transfected with the NACHO chaperone. A similar protocol was used to express α4-SEP and β2-pHuji in pcDNA3.1 with the control plasmid mTagBFP2 pcDNA3.1, as described in the Results section. Protect samples from light throughout the procedure to prevent photodestruction of the fluorophore.

  1. Prepare pH 7.4 imaging buffer (Table 1) and pH 5.5 quenching buffer (Table 2) as described23.

  2. Prewarm reagents to 37 °C, clean with 70% ethanol, and transfer to the biosafety cabinet. Remove the transfected cells from the incubator, clean with 70% ethanol, and place them in the biosafety cabinet.

  3. Remove the old medium by suction. Rinse the dish with 2 mL PBS for 3x, 10 min each at room temperature. Add 2 mL of pH 7.4 imaging buffer.

  4. Bring the dish to the confocal microscope. Insert the imaging dish with pH 7.4 imaging buffer into the slide holder and place the holder into the prewarmed environmental chamber on the confocal microscope.

  5. Connect the peristaltic pump tubing to the imaging dish holder and close the chamber.

  6. Open the imaging software and select lasers for the fluorophores of interest (pHuji 566/598 nm, α4-SEP 499/520 nm, β2-pHuji 568/603 nm, and mTagBFP2 359/461 nm).

  7. Scan the dish to locate cells for imaging using a 10x-60x objective. Increase magnification if desired and adjust focus depth as needed.

  8. Adjust laser intensity and detector sensitivity to visualize the fluorescence without a background signal. Adjust the aperture to increase the fluorescence signal, if required.

  9. Select the preferred resolution and capture the single image using the desired objective (e.g., 60x).

  10. Record the location on the dish map to relocate the same cells following the buffer change to pH 5.5 quenching buffer.

  11. Repeat the imaging process for several areas on the dish, recording each new location. Use the same capture settings for all images (experimental and control groups) to enable valid comparisons.

  12. Keeping only the outlet line connected to the peristaltic pump, slowly suction out the pH 7.4 buffer from the dish into a waste container.

  13. When most of the buffer is removed (without drying cells), transfer the inlet line to the pH 5.5 quenching buffer.

  14. Turn on the peristaltic pump to add pH 5.5 buffer slowly (1.5 mL/min) to the dish, ensuring cells remain attached.

  15. Run the pump for 20 min, observing that the buffer flows correctly into and out of the dish.

  16. Turn off the pump and incubate the cells with the pH 5.5 quenching buffer for an additional 20 min to allow equilibration.

  17. To repeat imaging, load the saved locations from the previous session to return to the same regions.

  18. Confirm that focal depth and position are correct, as they may shift after buffer exchange.

  19. Repeat imaging at all saved locations using the pH-quenched condition.

Table 1: Imaging buffer recipe.

The following reagents were added together, and the solution was adjusted to pH 7.4.

Reagent Concentration (mM)
NaCl 135
KCl 5
MgCl2 1.8
HEPES 20
d-glucose 1
Adjust solution to pH 7.4
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Table 2: Quenching buffer recipe.

The following reagents were mixed together, and the resulting solution was adjusted to a pH of 5.5.

Reagent Concentration (mM)
NaCl 135
KCl 5
MgCl2 0.4
CaCl2 1.8
MES 20
d-glucose 1
Adjust solution to pH 5.5
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4. Image quantification

NOTE: This procedure explains how to analyze cell images using ImageJ (National Institutes of Health, Bethesda, MD) and the corrected total cell fluorescence (CTCF) method32.

  1. Open ImageJ and open the image to be analyzed. The image will appear as a stack with each captured channel separated within the same frame.

  2. Navigate to the Phase channel. If the image is overly bright (common for blank or background regions), select Process > Enhance Contrast, then click OK.

  3. Using the magnifying glass tool, zoom in (+) on one cell. Using the Freehand selection tool, trace the outline of the cell in the Phase channel.

  4. Switch to the fluorescent channel corresponding to the fluorophore of interest. Set up the desired measurements by selecting Analyze > Set Measurements. Ensure that Area, Mean Gray Value, and Integrated Density are checked.

  5. To measure the selected region, choose Analyze > Measure or press Command + M (Mac) / Ctrl + M (Windows).

  6. Take three background measurements by tracing empty areas near the cell. Repeat steps for the desired number of cells.

  7. Measurement data will appear in a separate Results window in ImageJ. Copy and paste these data into Excel for analysis.

  8. Calculate the average background mean from the three background measurements.

  9. Compute the CTCF value for each cell using the following formula:
    CTCF=Integrated density−Area of cell×Mean background fluorescence

  10. Repeat this process for multiple cells and images for each experimental group (and pH condition, if applicable). Export all results to a data analysis software for statistical processing.

Representative Results

Following the above transfection procedures, α7 nAChRs and the NACHO chaperone protein were co-expressed in N2a cells. Both non-transfected and transfected cells were labeled with αBTX-AF647 24 h post-DNA transfection. Because αBTX is not membrane-permeable, all fluorescence detected originates from receptors present on the plasma membrane. In Figure 2A,A′, weak αBTX-AF647 staining was observed in non-transfected N2a cells, as expected. N2a cells endogenously express a small amount of α7 nAChRs, accounting for this minimal labeling. In contrast, positively transfected cells showed robust αBTX-AF647 labeling (Figure 2B,B′). The fluorescent signal appears as discrete puncta often associated with plasma membrane expression29. It is well known that nAChRs, when bound to ligands, can undergo endocytosis into recycling endosomes. Therefore, some internal labeling likely represents receptors that were initially located on the plasma membrane and subsequently internalized. Quantification using the CTCF method, as described in step 4, confirmed that cells transfected with α7 nAChR DNA exhibited significantly higher plasma membrane expression compared with untransfected controls (Figure 2C).

Figure 2: αBTX-AF647 primarily labels cells expressing the α7 nAChR on the plasma membrane.

Figure 2:

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Non-transfected and α7 nAChR-transfected N2a cells were treated with 80 nM αBTX-AF647 for 24 h prior to live-cell confocal imaging, according to steps 1 and 2. Images were captured at 60x magnification with a laser scanning confocal microscope. (A) αBTX-AF647 staining of endogenous α7 nAChRs expressed in non-transfected N2a cells. (B) α7 nAChR-transfected N2a cells labeled with αBTX-AF647. (A’, B’) Same as A and B with the removal of the phase channel. (C) CTCF analysis of αBTX-AF647 labeling of transfected and non-transfected N2a cells, as described in step 4 (****p < 0.0001, two-tailed unpaired t-test). Images shown are representative of four separate experiments per group. CTCF data are mean ± S.D. (N = 4, n = 60 for non-transfected cells, and 120 for +α7 nAChR transfected cells). This figure has been modified from26.

To help determine the relative distribution of cell-surface and internal receptors, an α7-pHuji recombinant construct was generated. This receptor shows strong plasma membrane expression when co-transfected with NACHO. To quantify the proportion of plasma membrane (external) receptors, N2a cells expressing α7-pHuji were imaged at pH 7.4 to measure the total fluorescence from all nAChRs (Figure 3A,A′). Because pHuji is a pH-sensitive fluorescent probe quenched at pH 5.5, this property was used to eliminate fluorescence from plasma membrane-expressed receptors, leaving only fluorescence from α7-pHuji subunits within the cells (Figure 3B,B′). By calculating the difference between total and internal fluorescence, the signal corresponding to α7-pHuji receptors localized on the plasma membrane was determined (Figure 3C,C′). Cells were analyzed using the CTCF method to quantify the proportion of internal and external receptors. Using this approach, 82.6% ± 3.6% of α7-pHuji receptors were localized on the plasma membrane, while 17.4% ± 3.6% remained intracellular (Figure 3D). Furthermore, pHuji fluorescence was restored after returning to the pH 7.4 imaging buffer, confirming reversible quenching of the fluorophore (Figure 3E,E′).

Figure 3: α7-pHuji nAChR transfection of N2a cells confirms localization of receptors at the cell plasma membrane.

Figure 3:

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N2a cells were transfected with α7-pHuji nAChR plasmid DNA as described in step 1 and live-cell imaged with buffer changes as described in step 3, to observe the location of the tagged receptors. Please note that receptors in organelles where the probe is positioned in acidic (quenching) environments were not captured. (A) N2a cells transfected with α7-pHuji and NACHO plasmid DNAs were imaged in pH 7.4 buffer. (B) The same N2a cells were imaged in a pH 5.5 buffer, showing only intracellular receptors. (C) Subtraction images of (A) and (B), representing α7-pHuji receptors expressed on the plasma membrane. (D) CTCF analysis of α7-pHuji nAChR-transfected N2a cells at pH 7.4 and pH 5.5, as described in step 4, shows that the majority of α7-pHuji nAChRs are localized to the plasma membrane of N2a cells. Data are mean ± S.D. (N = 3, n = 90 cells, ****p < 0.0001, unpaired two-tailed t-test). (E) The same N2a cells were imaged at a restored pH 7.4, verifying that changes in fluorescence are based on pH quenching. (A’, B’, C’, and E’) Same as A, B, C, and E without the phase channel. This figure has been modified from26.

The α4β2 nAChR subtype is also readily expressed in N2a cells. Using the above protocol and recombinant α4-SEP and β2-pHuji subunits, the relative location of each protein can be determined. Robust fluorescence is seen for each tagged subunit at a pH of 7.4 (Figure 4A–D), and is largely quenched at pH 5.5 (Figure 4E–H). The remaining fluorescence observed at pH 5.5 is from the tagged subunits located inside the cell in neutral pH environments. Bleed-through from each fluorescent protein was not observed (data not shown). Quantification of the fluorescence for each fluorescent tag shows that 23.4% ± 2.4 % of the α4-SEP are inside the cell, but the majority (76.5% ± 2.4 %) of the subunits are located on the plasma membrane (Figure 4I). The β2 subunit is known to have an endoplasmic reticulum retention sequence, resulting in less surface expression than the α4 subunit33. A similar expression pattern of β2-pHuji has been observed using the described procedures. The β2-pHuji subunit exhibits reduced trafficking to the plasma membrane, as evident by the data demonstrating that 43.5% ± 1.7% remain inside, while 56.5% ± 1.7% are expressed on the plasma membrane.

Figure 4: Double transfection of α4-SEP and β2-pHuji subunit DNAs results in robust plasma membrane expression.

Figure 4:

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Cells were subcultured onto poly-D-lysine-coated plates and transiently transfected with α4-SEP and β2-pHuji as described in step 1 and imaged following step 3 procedures. Images were acquired at 60x magnification using a laser scanning confocal microscope at excitation/emission wavelengths of 499/520 and 568/603 for α4-SEP and β2-pHuji, respectively. (A - E) Cells were imaged with an initial pH 7.4 imaging buffer. (F - J) Following, the buffer was exchanged to the 5.5 quenching imaging buffer. (I) Expression of internal and external α4-SEP and B2-pHuji subunits was quantified, and both α4-SEP and β2-pHuji subunits exhibited significantly more external than internal subunits (α4-SEP: external = 76.5% ± 2.4%, internal = 23.8% ± 2.4%, β2-pHuji external = 56.5% ± 1.7%, internal = 43.5% ± 1.7%, N = 3, n = 51 cells. One-way ANOVA, Tukey’s post-hoc analysis ****p < 0.0001 and *** p = 0.0003).

To demonstrate that the presented protocol can be extended to include an additional plasmid, N2a cells were triple-transfected with α4-SEP, β2-pHuji, and the control mTagBFP2 DNAs. mTagBFP2 is a fluorescent protein that is brighter and more photostable than mTagBFP and is not pH-sensitive34. Because mTagBFP2 was not fused to another gene sequence, the protein was localized to the cytosol of the transfected cells (Figure 5B,G). Images were acquired 24 h post-transfection using both pH 7.4 and pH 5.5 imaging buffers to demonstrate that this method yields high transfection efficiencies and robust plasma membrane expression of nAChR subunits (Figure 5A–E). Quantification of individual cells that were triple-transfected showed results similar to those in Figure 4: 17.2% ± 3.7% of α4-SEP was localized internally, whereas 82.8% ± 3.7% was found on the plasma membrane (Figure 5K). As noted in the preceding paragraph, the β2 subunit traffics less efficiently to the cell surface, which was again observed in triple-transfected cells, with 36.6% ± 11.7% located intracellularly and 63.4% ± 11.7% on the plasma membrane.

Figure 5: α4β2 nAChRs express well on the plasma membrane when co-transfected with a chaperone mimic, mTAGBFP2, DNA.

Figure 5:

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Cells were subcultured using the same protocol as Figure 4 and transiently transfected using equal amounts of endotoxin-free α4-SEP, β2-pHuji, and mTAGBFP2 DNAs 24h prior to imaging. Images were captured at 60x magnification with a laser scanning confocal microscope at excitation/emission wavelengths of 499/520, 568/603, and 359/461, for α4-SEP, β2-pHuji, and mTAGBFP2, respectively. Cells were imaged in photoimaging buffer at (A-E) pH 7.4 and (F-J) pH 5.5. (I) Expression of α4-SEP and β2-pHuji subunits were quantified and were found to have significantly more subunits expressed on the plasma membrane than those that remained intracellularly (α4-SEP: external = 82.8% ± 3.7%, internal = 17.2% ± 3.7%, β2-pHuji: external = 63.4% ± 11.7%, internal = 36.6% ± 11.7%, N = 3, n = 71 cells. One-way ANOVA, Tukey’s post-hoc analysis ****p < 0.0001 and *p = 0.0219).

Discussion

The methodologies presented for expressing and quantifying nAChR subunits in mammalian N2a cells address long-standing challenges in achieving rapid (~24 h) and abundant nAChR (> 60%) plasma membrane expression for drug discovery, localization, and trafficking studies. A key strength of this set of protocols is its adaptability for both homomeric (α7) and heteromeric (α4β2) nAChRs, and it is likely applicable to other nAChR subtypes, along with the co-expression of chaperone proteins. By leveraging pH-sensitive tags such as SEP and pHuji, the approach enables live-cell discrimination and quantification between plasma membrane and intracellular populations of nAChRs. This distinction is crucial given that nAChR subtypes are trafficked by a variety of chaperone and auxiliary proteins, including 14-3-3, NACHO, RIC-3, and lynx-1 proteins, and by ligands, like nicotine17,18,35,36,37,38,39. The use of pH-sensitive fluorescent reporters allows for the identification of receptor location in live cells under different imaging conditions, without the need for chemical fixation. CTCF analysis adds quantitative rigor, enabling reliable comparison of surface and internal receptor expression under various experimental conditions32.

The protocol’s transfection strategy, which relies on a DNA transfection reagent for lipid-based DNA delivery, provides high efficiency in N2a cells and can be easily adapted for additional subunits or accessory proteins. There is much to learn about how chaperone and auxiliary proteins traffic heteromeric nAChRs, including the α4β2 subtype, to the plasma membrane. α4β2 nAChRs are the majority of expressed central nervous system (CNS) nAChRs40,41, and occur in two distinct stoichiometric isoforms, (α4β2)2β2 and (α4β2)2α4, with high and low sensitivity to agonist (HS and LS, respectively)42,43,44,45,46,47,48. Endogenous proteins, like 14-3-3 and lynx1, regulate isoform expression by altering receptor trafficking or reducing PM receptor turnover37,38. As additional chaperone and auxiliary proteins that target nAChRs are identified, it will be advantageous to express these proteins in mammalian cell lines for the study of dynamic nAChR trafficking events. These procedures are also applicable to current or new existing pharmacological agents, as well as disease-relevant mutations on nAChR localization and turnover. By means of live-cell imaging, receptor locations can be tracked in real time, allowing for valuable insights into trafficking pathways.

Potential limitations include the reliance on overexpression systems, which may not fully recapitulate endogenous receptor regulation or trafficking dynamics found in neurons or other cells in vivo. The addition of C-terminal fluorescent tags, while highly informative, could potentially affect receptor folding, assembly, or interactions with cellular partners. However, adding these types of tags to nAChR subunits has previously been demonstrated to show that the receptors still respond to acetylcholine29,49. An alternative to using pH-sensitive probes is to use HA tags and antibodies to only label nAChRs on the plasma membrane17. However, using this approach, receptors that are internalized during the incubation time will be labeled, and information regarding the internal receptor population is lost. Additional time is also needed to fix and stain the cells before imaging. Another limitation is that information regarding the location of nAChRs in organelles with an acidic pH (< 7.0) cannot be quantified using pH-sensitive probes attached to the C-terminal of the subunits. nAChR subunits are oriented in the endoplasmic reticulum (ER) and Golgi apparatus membranes with the extracellular domain and C-terminal positioned on the luminal side of the organelles. The ER pH is neutral (7.2 −7.4), while the Golgi intracellular pH is more acidic (≤ 6.7)50. For experiments that desire to capture the movement of the nAChRs through the Golgi, the pH-sensitive probe could be added to the intracellular M3-M4 loop. However, as many chaperone or auxiliary proteins interact within the intracellular loop, the addition of a bulky fluorescent protein may cripple these interactions. Alternative approaches could also be the use of a non-pH sensitive probe (attached to the C-terminal) coupled with organelle-specific labels to detect changes in nAChR movement through intracellular acidic compartments29,38.

The presented protocols are positioned to advance investigations into the molecular mechanisms underlying nAChR expression on the plasma membrane and pharmacological modulation. Its flexibility supports the incorporation of auxiliary proteins, implementation of disease-relevant mutations, and application of labeled ligands or live-cell pharmacology. Overall, the described workflow offers a robust platform for dissecting the cell biology of nAChRs and their modulation by chaperones, drugs, and disease processes. By enabling precise quantification of receptor localization at the plasma membrane versus internal (non-acidic) compartments, this method provides critical insights for both basic neuroscience research and the development of therapeutics targeting nAChRs.

Acknowledgments

The authors would like to thank James Janoso for his technical microscopy expertise and the Center for Transformative Research in Metabolism (National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM130443) for supporting the Microscopy Core at the University of Alaska Fairbanks. LW’s efforts were partially supported by funding from the Higher Education Investment Fund, provided by the state of Alaska through the University of Alaska. SMS’s efforts were supported by the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395. Research reported in this publication was supported by the National Institute of Mental Health of the National Institutes of Health (MMW by award number R03MH135358) and the National Institute of General Medical Sciences (MMW by award number R16GM150455) of the National Institutes of Health.

Footnotes

A complete version of this article that includes the video component is available at http://dx.doi.org/10.3791/69371.

Disclosures

The authors have nothing to disclose.

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