Chapter 4
Chemiluminescence Assays to Investigate Membrane
The rate of internalisation can be examined by chemilumi-nescence-based assays. For these approaches it is necessary to have antibodies that bind to the external portions of the channel to be examined. This allows antibody binding to a pool of channels on the cell surface while the cells are held in a state that is non-permissive to trafficking, achieved by cooling the cells to 4°C.
When the cells are warmed back to temperatures permissive to protein trafficking, the channel/antibody complex is endocytosed from the cell surface. At an appropriate time, internalisation is halted by fixation and the proportion of labelled channels on the cell surface can be examined. Surface exposed channels are labelled with horseradish peroxidised (HRP)-conjugated antibodies allowing the use of a luminescence-based assay.
1. Culture medium: Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum.
2. Culture plates: Sterile 24-well culture plates.
3. HEK cells stably expressing KATP channels (SUR1 and Kir6.2 bearing an extracellular HA-epitope – KATP-HA) see ref. 7.
4. Transfection reagent: Fugene6 (Roche Diagnostics).
5. Phosphate buffered saline (PBS): 10-mM phosphate buffer, 2.7-mM KCl, 137-mM NaCl prepared from tablets (Sigma) by dissolving one tablet in 200-ml of water.
6. Antibodies targeted against external epitope: Rat anti-HA (clone 3F10) (Roche Diagnostics) (see Note 1).
7. Labelling medium: DMEM supplemented with 5% goat serum freshly prepared before each experiment.
8. HRP-conjugated antibodies: Anti-rat HRP-conjugated anti-bodies (Sigma).
9. Cell fixing solution: prepare 2% fresh paraformaldehyde (w/v) solution in PBS for each experiment. The solution should be carefully heated in a fume-hood to dissolve and then cooled to room temperature before use.
10. Cell solubilisation solution − 2% (w/v) sodium deoxycholate in 1× PBS.
11. Sensitive enhanced chemiluminescence (ECL) reagent (Pierce).
12. Automated microplate reader: e.g. POLARstar OPTIMA (BMG labtech) although other companies offer other options.
4.2. Materials
4.2.1.
Chemiluminescence Techniques
13. Modulators of endocytosis: Mammalian expression plasmids containing genes that disrupt clathrin-mediated endocytosis (e.g. dominant-negative (DN) forms of dynamin or µ2).
1. BCA reagent: Mix 49 parts bicinchoninic acid solution (Sigma) and 1 part 4% (w/v) copper sulphate solution immediately before use.
2. Protein standards: Several samples of known protein con-centration prepared by diluting 10-mg/ml BSA in the same buffer used to solubilise cells. The concentration of the pro-tein standards should range from 0 to 10 µg/ml.
3. Cell lysates: Prepared as described in the individual proto-cols.
The following protocol allows the determination of the rate of endocytosis of a channel from the cell surface. The internalisation of channels is investigated over a time course with samples taken at regular intervals. Each time-point is required to be performed in duplicate and therefore requires two wells of cells. A typical experiment will consist of at least five time-points (i.e. at least ten wells). An example of the data obtained form this type of experi-ment is shown in Fig. 4.1a.
1. Cells expressing KATP-HA are cultured to confluence in a 24-well culture plate under experimental conditions (see Note 2). Two wells are required for each time-point to be examined. In parallel, culture cells which do not express KATP-HA (untransfected) in a similar manner are to serve as negative controls.
2. Remove the culture medium and cool the cells to 4°C. Rinse three times with chilled PBS to remove the residual medium.
3. Overlay the cells in each well with 150 µl of chilled (4°C) label-ling medium containing 0.2 µg/ml rat anti-HA antibodies and incubate at 4°C for 1 h to label channels on the cell surface.
4. Remove the labelling medium and rinse the cells six times with chilled PBS to remove any unbound antibodies.
5. Fix the cells in two of the wells by adding 100 µl of cell fixing solution. Incubate for 10 min at 4°C. These samples serve as zero time-point controls to which the extent of internalisa-tion at other time-points will be compared.
6. Incubate the cells in pre-warmed (37°C) labelling medium containing no antibodies at 37°C for varying lengths of time to allow channel internalisation (see Note 3).
4.2.2. Cell Lysate Protein Determination Assay
4.3. Methods
4.3.1. Endocytosis Kinetics by
Chemiluminescence
7. Fix the cells by adding 100 µl of cell fixing solution. Incu-bate for 10 min at 4°C.
8. Rinse the cells three times with chilled PBS to remove resid-ual fixative.
9. Incubate the fixed cells with labelling medium containing 1.2-µg/ml anti-rat HRP-conjugated antibodies at room temperature for 1 h.
10. Rinse eight times with PBS to remove any unbound anti-bodies.
11. Solubilise the cells by incubating with 150 µl of cell solubili-sation solution for 20 min with gentle agitation.
12. Pipette two 50-µl aliquots of each solubilised cell sample into individual wells of a clear, flat-bottomed black-walled 96-well plate. Retain the remaining 50-µl sample on ice for future use.
13. Assay the HRP content of each lysate by adding 50 µl of high-sensitivity ECL reagent. Use an automated microplate reader to determine the luminescence. Allow the lumines-cence signal to reach a steady state, which typically takes between 5 and 10 s following addition of the ECL reagent.
14. Determine the total protein content of each lysate using the remaining lysate from step 10 (see Subheading 4.3.3).
15. Correct the luminescence readings for each sample relative to protein content (relative fluorescence units/µg protein).
16. Deduct the mean corrected luminescence readings for the untransfected control cells (which correspond to the background signals) from each of the luminescence readings for each test sample. This results in two background subtracted Fig. 4.1. Endocytosis and cell surface density of KATP channels in HEK293 cells. A time course of internalisation (a) and the steady-state surface expression (b) of KATP channels in HEK293 cells investigated by chemiluminescence-based methods. In (b) the surface density is increased by co-expression of dominant-negative (DN) forms of dynamin 1 (Dyn1), dymanin 2 (Dyn2) and µ2, each of which prevents clathrin-mediated endocytosis. (Reproduced from ref. 6 with permission from the Nature publishing group).
readings for each lysate which is in turn one of two samples from the original 24-well culture plate.
17. Normalise each of the corrected readings to the signal at zero time, i.e. no internalisation. Perform the experiment in triplicate and express as mean ± SEM and plot vs. internalisa-tion time. An example of the data is shown in Fig. 4.1a.
The above protocol (see Subheading 4.3.1) may be modified to allow the determination of the relative steady-state surface expression of channels at a given time. The protocol is identi-cal except for the omission of steps 5 and 6 (internalisation of labelled channels at 37°C). The channels on the cell surface are labelled at 4°C, at which temperature no trafficking will occur, and then the cells are fixed. The chemiluminescence protocol then continues and the channel cell surface density is quantified.
The process of clathrin-mediated endocytosis may be disrupted by the expression of genetically encoded modulators (e.g. DN forms of dynamin 1, dynamin 2 and µ2). An example of the data is shown in Fig. 4.1b.
This protocol describes how the protein content of cell lysates is to be determined using the BCA method. Many alternative methods are available in kit form from numerous suppliers.
1. Pipette 10 µl of each cell lysate into individual wells of a flat-bottomed 96-well plate in duplicate.
2. Pipette 10 µl of each protein standard into individual wells of a flat-bottomed 96-well plate in triplicate.
3. Add 200 µl of BCA reagent to each well and incubate the plate at 37°C for 30 min.
4. Measure the absorbance of each sample at 550 nm using a plate reader.
5. Plot the mean absorbance values of the protein standards against protein concentration to create a calibration curve from which the mean protein content of the cell lysates can be extrapolated.
1. Antibodies targeted against extracellular regions of KATP are not commercially available. Therefore an extracellular HA-epitope was inserted into an extracellular loop of the Kir6.2 subunit using PCR-based techniques. It is vital that the insertion of epitopes is shown not to interfere with normal channel functions. The presence of extracellular HA 4.3.2. Steady-State
Cell Surface Density Chemiluminescence-Based Assay
4.3.3. Cell Lysate Total Protein Assay
4.4. Notes
epitopes allows the use of well-characterised commercial anti-HA antibodies. It is important that the antibodies used contain no preservatives (e.g. sodium azide or thermosial) as these may harm the cells being investigated.
2. The adherence of cells may be improved by coating the bot-tom of each well with 0.0001% poly-L-lysine (>70,000 KDa) solution for 1 h. Each well should be rinsed with 1× PBS before seeding the cells into them.
3. The duration of the internalisation steps required will vary between different ion channels and should be determined empirically. For most applications 0, 15, 30 and 60 min should prove to be a good starting point.
The authors would like to thank Dr. Jamel Mankouri for the development of the chemiluminescence-based assays.