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

Understanding nicotinic acetylcholine receptor trafficking to mammalian plasma membranes is crucial for investigating chaperone-mediated regulation and evaluating target-selective therapeutics. A primary advantage of pH sensitive probes combined with live cell confocal microscopy is the high spatial resolution of subunit location. To begin, prewarm reagents to 37 degrees Celsius.

Thaw the nAChR subunit DNase completely and bring the transfection reagent to room temperature. Clean all reagents with 70%ethanol before placing them inside the biosafety cabinet. To express alpha-7 nAChRs, in a micro centrifuge tube, dilute alpha-7 and NACHO plasmid DNase into reduced serum medium.

Mix gently by pipetting after DNA addition. Alternatively, to express alpha-4 beta-2 receptors, dilute 4 micrograms of each subunit DNA into a total of 250 microliters of reduced serum medium and mix gently. Adjust the subunit ratios to optimize expression of specific receptor isoforms.

For alpha-4 beta-2 receptors with an additional plasmid, add equal amounts of each plasmid DNA. Next, in a new micro centrifuge tube, dilute 8 microliters of transfection reagent dropwise into 250 microliters of reduced serum medium. After a 5 minute incubation at room temperature, combine the diluted DNase with the diluted transfection reagent dropwise into a tube.

Mix gently by pipetting and incubate for 20 minutes at room temperature. While complexes form, replace the medium in the culture dishes with 1 milliliter of serum-free EMEM. Add 500 microliters of the DNA transfection reagent complex dropwise to each dish.

Mix gently by rocking the plate to distribute evenly, especially over the glass bottom surface. Incubate the plate at 37 degrees Celsius for 24 hours and replace the medium after 4-6 hours. Remove the old medium by suction.

Rinse the dish three times with two milliliters of PBS for 10 minutes each at room temperature. Then add 2 milliliters of pH 7.4 imaging buffer to the dish. Insert the imaging dish into the slide holder of a confocal microscope and place the holder into the pre-warmed environmental chamber.

Connect the peristaltic pump tubing to the imaging dish holder and close the environmental chamber. Now, launch the imaging software and select lasers for the fluorophores of interest. Scan the dish to locate cells for imaging using a 10-60 x objective.

Increase magnification if desired, and adjust the focus depth as needed. Select the preferred resolution and capture a single image using the desired objective. Record the location on the dish map to relocate the same cells after buffer change.

Repeat imaging for several areas on the dish using the same capture settings for all images. 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. Then transfer the inlet line to the pH 5.5 quenching buffer.

Turn on the peristaltic pump to add pH 5.5 buffer at a flow rate of 1.5 milliliters per minute, ensuring that the cells remain attached. After 20 minutes, turn off the pump and incubate the cells in the buffer for an additional 20 minutes to allow equilibration. After incubation, repeat imaging of the saved locations.

To begin image quantification, open ImageJ and load the image to be analyzed. The image will appear as a stack with each captured channel displayed in the same frame. Navigate to the phase channel.

If the image appears overly bright, select Process and choose Enhance Contrast then click OK.Use the magnifying glass tool to zoom in on one cell. Use the freehand selection tool to trace the outline of the cell in the phase channel. Then switch to the fluorescent channel that corresponds to the fluorophore of interest.

Set the desired measurements by selecting Analyze and clicking on Set Measurements. Ensure that Area, Mean gray value, and Integrated density are checked. To measure the selected region, choose Analyze and then click Measure.

Take three background measurements by tracing empty areas near the selected cell. After performing measurements for all desired cells, copy and paste the measurement data into Excel for analysis. Then calculate the average background mean from the three background measurements.

Compute the corrected total cell fluorescence or CTCF value for each cell using the provided formula. Using the same workflow, analyze images acquired after buffer change. Export all results to data analysis software for statistical processing.

Weak alpha-BTX AF647 staining was observed in non-transfected N2a cells while robust punctate labeling was detected in cells transfected with alpha-7 DNA. Quantification confirmed that plasma membrane expression of alpha-7 nAChRs was significantly higher in alpha-7 transfected N2a cells than in non-transfected controls. At pH 7.4, total alpha-7 pHuji fluorescence was visible at the plasma membrane.

in transfected N2a cells. At pH 5.5, alpha-7 pHuji fluorescence was reduced, revealing internal receptor localization. The external alpha-7 pHuji receptor fluorescence accounted for the majority of receptor expression.

Quantification showed that 82.6%of alpha-7 pHuji receptors were located on the plasma membrane, while 17.4%were internal. Fluorescence was restored upon return to pH 7.4, confirming pHuji signal reversibility. At pH 7.4, N2a cells expressing alpha-4 SEP and beta-2 pHuji showed robust membrane fluorescence for both subunits.

At pH 5.5, fluorescence was quenched, indicating internal localization of some alpha-4 SEP and beta-2 pHuji subunits. Quantification showed that 76.5%of alpha-4 SEP was at the plasma membrane, and 23.4%was internal. In triple transfected cells, alpha-4 SEP displayed 82.8%membrane localization and 17.2%internal expression.

In the same cells, 63.4%of beta-2 pHuji subunits were localized on the membrane while 36.6%were located intracellularly. Using the presented protocol, we characterized novel chaperone effects on nicotinic receptor expression and trafficking, and evaluate new target-selective therapeutics. An advantage of our protocol is adaptability for homomeric and heteromeric nicotinic receptors, enabling quantification of relative cellular location.

Our approach enables rapid robust nicotinic receptor plasma membrane expression, aiding chaperone-mediated trafficking analysis and drug discovery efforts.