Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

1. Preparation of cover slips

NOTE: These steps must take place in a sterile tissue-culture hood. Quantities are given for the preparation of 10 dishes.

1. Place ten autoclaved, 30 mm borosilicate cover glass slips individually into ten 35 mm non-treated sterile dishes and rinse once with 2 mL of sterile, distilled water.
2. Dilute 1 mL of 0.1% w/v poly-_L-lysine solution into sterile, distilled water to a total volume of 10 mL (final concentration of 0.01% w/v). Mix well, then pipette 1 mL onto each cover slip and incubate at room temperature for 20 min.
3. Aspirate the poly-_L-lysine and wash each cover slip twice with at least 2 mL of sterile distilled water. Leave until completely dry i.e., at least 3 h.

2. Seeding HEK-293T cells

NOTE: These steps must take place in a tissue-culture hood. HEK-293T cells were chosen for their low current background and ease of growing in culture. This protocol may be adapted to other cell types.

1. Rinse an 80-90% confluent T75 flask of HEK-293T cells once with 12 mL phosphate buffered saline (PBS) before incubating with 2 mL trypsin for 2-5 min, or until the cells are fully detached and almost completely dissociated.
2. Resuspend the cells by adding 10 mL Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. Pipette gently against the bottom of the flask to break up remaining clumps of cells.
3. Add 2 mL of supplemented DMEM to the desired number of 35 mm dishes containing coated cover slips. Add 100 µL of resuspended cells to each dish. Incubate overnight at 37 °C.

3. Transfection

NOTE: These steps must take place in a tissue-culture hood. Quantities are given for the transfection of 10 dishes. For site-specific ANAP incorporation, the DNA codon at the position intended for labeling must be replaced with the amber (TAG) stop codon. This construct is co-transfected with two plasmids: pANAP and peRF1-E55D^12,15. pANAP encodes several copies of an ANAP-specific tRNA/tRNA synthetase pair. In the presence of ANAP, transfection of this plasmid produces tRNA charged with ANAP that recognizes the amber stop codon. peRF1-E55D encodes a dominant negative ribosomal release factor that increases the yield of full-length, ANAP-tagged protein.

1. Prepare a 1.5 mL tube with 10 µg pANAP, 10 µg peRF1-E55D, and DNA for the construct intended for labeling with ANAP. Bring to a final volume of 500 μL with unsupplemented DMEM.
2. In a separate tube, prepare 3 µL of lipid-based transfection reagent (see Table of Materials) for each 1 µg of DNA and bring to a final volume of 500 μL with unsupplemented DMEM.
3. Combine the DNA and transfection reagent mixtures in a single tube and incubate for 20 min at room temperature.
4. Add 400 µL of the 1 mM ANAP stock (trifluoroacetate salt in 30 mM NaOH) to 20 mL supplemented DMEM for a final concentration of 20 µM ANAP. Replace the old media from the plated cells with 2 mL of the ANAP-containing media per dish.
5. Pipette 10% of the DNA transfection mix onto each dish. Incubate at 33 °C for 2-4 days before experiments. Incubation at 33 °C slows the cell division and increases the protein yield per cell¹⁶.

4. Unroofed membrane experiments

1. Use a pair of forceps to break a cover slip with transfected cells into smaller fragments.
2. Follow one of the procedures below to unroof cells.
   1. If using pre-coated cover slips, rinse a fragment with PBS, then place it on the bottom of a 35 mm dish containing 2 mL PBS. Briefly sonicate using a probe sonicator (50 W, 20%-40% amplitude, 3 mm probe) positioned 3-5 mm above the sample to unroof cells and leave behind adherent plasma membrane fragments (Figure 2A,C).
      NOTE: Sonicator power, duration, and probe height above the sample can all be varied to obtain a high yield of unroofed membranes without completely denuding the coverslip.
   2. If not using pre-coated cover slips, rinse a cover slip fragment with PBS, then dip into a tube containing 0.1% w/v poly-_L-lysine for ~30 s before briefly sonicating (as in step 4.2.1) to unroof cells and leave behind unroofed/partially unroofed plasma membrane fragments (Figure 2A,C,D). Brief exposures to poly-_L-lysine have been demonstrated to improve adherence to the coverslip¹³.
3. Place the sonicated fragment into a cover glass-bottom 35 mm dish containing 2 mL bath solution and mount onto an inverted microscope equipped with a high NA, 60x water immersion objective. The camera port of the microscope is connected to a spectrograph in series with a high sensitivity CCD camera. Perfuse the bath chamber (0.5 – 1 mL/min) with buffer using a peristaltic pump. The composition of the buffer will vary depending on the protein under study.
   NOTE: If the user does not have access to an objective with a long working distance, it may be impossible to focus on the unroofed membrane fragments due to the extra height of the cover slip. An alternative is to seed cells directly onto dishes with poly-_L-lysine glass bottoms (see Table of Materials for an example). This will also reduce potential aberrations in the image associated with focusing through two pieces of glass. These aberrations do not affect the shape of the spectra acquired.
4. Identify unroofed membrane fragments expressing the ANAP-labeled channel by looking for channel fluorescence (Figure 2C,D).
   NOTE: It is recommended to use an additional fluorescent label (where the emission spectrum is distinguishable from the ANAP emission spectrum) to help identify unroofed membranes containing the protein of interest. The experiments in Figure 2C,D were performed on ANAP-labeled channels with C-terminal fluorescent protein tags.
5. Partially engage the spectrometer mask (raise ~10%) between the camera port on the microscope and the spectrograph. The shadow of the mask will appear on the camera image. Align the unroofed membrane with the spectrometer mask, by adjusting the microscope stage. Acquire a bright field and fluorescence image of the unroofed membrane. These will be used to select a region of interest for analysis.
6. Bring the tip of the microvolume perfusion system close to the unroofed membrane.
   NOTE: To reduce the background fluorescence, the outflow of the perfusion system was replaced with a custom tip made from borosilicate glass.
7. To image fluorescence spectra, excite the membrane with a 385 nm LED through a 390/18 nm band-pass excitation filter and a 416 nm edge dichroic. Collect emitted light through a 400 nm long-pass emission filter (Figure 2B).
8. Engage the spectrometer mask and ensure the emitted light is passed through. Engage the spectrometer gratings (300 grooves/mm). With the gratings in place, the light diffracted by the spectrometer will be projected onto the chip of the CCD camera to produce spectral images (Figure 3A). These images retain spatial information in the y dimension. The x dimension is replaced with wavelength.
9. Optionally, if the protein of interest is tagged with a fluorescent protein, acquire a spectral image of the fluorescent protein using the appropriate filter set.
10. Take one or more 0.1-10 s exposures at the start of the experiment while perfusing nucleotide-free buffer solution. These will be used to correct and normalize data throughout the rest of the experiment (see section 5 below).
    NOTE: Choice of exposure time will depend on the expression level achieved, the brightness of the fluorophore, and the optics. Exposure time should be chosen to maximize signal and minimize the observed bleaching rate. The exposure time range given in 4.10 is suitable for equilibrium binding measurements but may be useful for measuring slower kinetic changes¹⁰. The ability to use short exposure times to track faster kinetics will be limited by protein expression levels and photobleaching, rather than hardware.
11. Apply a range of concentrations of TNP-ATP (usually prepared in bath solution) to establish a concentration-response curve. Perfuse each solution for at least 1 min to ensure that a steady state is reached and wash out each concentration with bath solution for at least 1 min.
    NOTE: It is important to ensure that the perfusion system can rapidly reach equilibrium (Figure 2E) and achieve the correct local concentration of TNP-ATP (Figure 2F).
12. Take an exposure (with the same duration as used in step 4.10) at each concentration and at the end of each washout.

5. Spectral analysis

NOTE: These instructions are written for use with the analysis code “pcf.m”, which can be found at GitHub. https://github.com/mpuljung/spectra-analysis¹⁰. Additional and alternative code can be found at https://github.com/smusher/KATP_paper_2019¹¹. We have described the operations performed by the software here so that the user may create their own code or choose to analyze the data manually.

1. Start the analysis program by typing the program’s name (“pcf”) in the command line.
2. When an open file/folder dialog box opens with the prompt: “Select files for ROI”, select the file names associated with the brightfield and fluorescence images of the unroofed membrane. A prompt will appear in the command line to type the name of the output file.
3. Type the file name and hit enter.
4. When the software displays the brightfield and fluorescence images, select a region of interest (ROI) in the spectral image corresponding to the location of the unroofed membrane fragment or excised patch (see section 6) following the software prompts. Select a background region in the same spectral image (representing the same wavelength range as in the ROI) corresponding to a section of cover slip or dish with no membrane attached (Figure 3A). The software will prompt to click the Top of the ROI and press Enter, click the bottom of the ROI and press Enter and then repeat this process for the background region.
5. When an open file/folder dialog box opens with the prompt: “Select File for FP Spectrum”, select the file name associated with the fluorescent protein (FP) spectrum (optional step 4.9). If no FP spectrum was acquired, select a different spectrum file. The FP spectrum serves as quality control to distinguish between tagged protein and background fluorescence.
6. When an open file/folder dialog box opens with the prompt: “Select Files for Analyisis”, select all the files corresponding to the ANAP spectra (from steps 4.10 to 4.12), including the files needed for bleach correction.
7. When an open file/folder dialog box opens with the prompt: “Select Files for Bleaching Collection”, select the subset of files from step 5.6 corresponding to initial spectra acquired in nucleotide-free solution at the beginning of the experiment or spectra acquired during washes in nucleotide-free solution to be used for correction (from steps 4.10 to 4.12).
8. Line-average each image to produce spectra, i.e., average the intensity for all pixels in the y dimension of an ROI or background region at each wavelength. (Figure 3B). Subtract the resulting averaged background spectrum from the averaged spectrum acquired from the ROI to remove background fluorescence and fluorescence from unbound TNP-ATP (Figure 3C). These steps are performed automatically by the software.
9. Determine the ANAP intensity for each exposure by averaging the intensity of a 5 nm window centered around the ANAP peak of the subtracted spectra (typically ~470 nm but may vary depending on the local microenvironment of the ANAP residue).
   NOTE: Figure 3D shows 6 spectra obtained from consecutive 10 s exposures of an unroofed membrane fragment expressing ANAP-tagged channels. The inset shows the averaged intensity of the peak of each spectrum. The software will automatically find the peak wavelength in the first spectrum acquired and use this value throughout. The intensity will be calculated automatically by the software.
10. Normalize the ANAP intensities for each experiment by dividing the ANAP intensity of a given exposure (F) by the ANAP intensity of the first exposure in the time series, which was taken in step 4.10 (F_max). Again, the software performs these calculations automatically.
11. Perform the steps below to obtain data.
    1. To correct for ANAP photobleaching, first fit a single exponential decay, (F/F_max) = A*exp(-t/τ)+(1-A), where t is the cumulative exposure time, τ is the time constant and A is the amplitude) to either the intermediate wash steps between TNP-ATP applications or to multiple initial exposures taken before washing on TNP-ATP (Figure 3D, inset).
       NOTE: The software will display this fit and prompt to accept or reject it. If the fit is rejected, another opportunity will be provided to select files for bleaching correction.
    2. Divide the normalized (in step 5.10) ANAP spectra by the predicted value of the exponential fit from step 5.11.1 at each time point (Figure 3E).
       NOTE: For the example shown, the observed normalized peak fluorescence at 50 s is 0.65 and the predicted fluorescence from the exponential fit is 0.64. To correct for bleaching, divide the observed value (0.65, Figure 3E inset, empty circle) by the predicted value (0.64, Figure 3E inset, dashed line) to produce the corrected value (~1, Figure 3E inset, colored circle). If bleaching correction is adequate, the intensity of ANAP from all exposures acquired in the absence of nucleotides should be approximately equal (Figure 3E). These calculations are performed automatically by the software.
    3. Obtain the output as an image plotting the data and a tabbed spreadsheet containing the raw spectra, subtracted spectra, spectra corrected for photobleaching, and the peak data for each file so that further analysis may be conducted.

6. Patch-clamp fluorometry experiments

1. Pull patch pipettes from thick-walled borosilicate glass capillaries to a resistance of 1.5 MΩ to 2.5 MΩ when filled with pipette solution. The composition of the pipette solution will vary depending on the protein under study.
2. Transfer a cover slip with transfected cells onto a cover glass-bottom 35 mm dish containing 2 mL bath solution and mount onto an inverted microscope equipped with a high NA, 60x water immersion objective. Perfuse the bath chamber (0.5 – 1 mL/min) with bath solution using a peristaltic pump. As for the pipette solution, the bath solution will vary depending on the protein under study.
3. Identify a cell expressing ANAP-labeled channels by looking for fluorescence at the cell membrane.
4. Fill a patch pipette with pipette solution. Apply gentle positive pressure to the pipette and place in the bath chamber. Press the pipette against the membrane of the cell and apply gentle suction to achieve a GΩ seal (Figure 4A).
5. Excise the patch by rapidly moving the pipette holder away from the cell (Figure 4A).
   NOTE: Excising the patch in this fashion should form an inside-out patch, with the cytosolic domains of the protein exposed to the perfusion system. If the location of the nucleotide binding site under study is not cytosolic, it will be necessary to use outside-out patches or whole-cell recordings to perform PCF experiments.
6. Bring the tip of the patch pipette close to the tip of the perfusion system, and check that the patch is within the slit of the spectrometer mask (Figure 4A).
7. Apply TNP-ATP and image spectra as in steps 4.10-4.12, while simultaneously recording the ionic current response to nucleotide application.
   NOTE: The pipette glass may introduce spatial aberrations and reflections in the acquired images. However, these aberrations will not affect the shape of the acquired spectra and reflected excitation light is easily separated from fluorescence using either the spectrograph or a long-pass emission filter.
8. Analyze the spectra. Spectra imaged from excised patches can exhibit over-subtraction of unbound TNP-ATP fluorescence due to the exclusion of TNP-ATP from the glass of the patch pipette (Figure 4C-E). This over-subtraction does not affect the ANAP emission spectrum and so can be ignored.
   NOTE: As the fluorescence signal in excised patches will be lower than in unroofed membranes, it is important to use an exposure time which gives high enough signal-to-noise without bleaching ANAP too rapidly.