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

As ATP is an important cellular messenger, we developed a method that allows us to measure ATP binding to its receptors with high precision. Using FRET between nucleotide derivatives and fluorescently tagged proteins, we can monitor nucleotide binding to intact functional receptors in real time and with excellent spatial resolution. We use this method to understand the effects of cellular metabolism on the ATP sensitive potassium channel, which is implicated in certain forms of diabetes.

To set up an unroofed membrane experiment, first use a pair of forceps to break a small fragment from a cover slip with transfected cells and rinse the fragment with PBS. If the cover slip is pre-coated with polylysine, place the fragment on the bottom of a 35 millimeter dish containing two milliliters of PBS and hold a three millimeter probe sonicator three to five millimeters above the sample, pulsing at 50 watts and 20 to 40%amplitude for 200 to 500 milliseconds to unroof the cells. If the cover slips are not pre-coated, after rinsing with PBS, dip the fragment into a tube containing 0.1%poly-L-lysine for about 30 seconds before unroofing the cells with brief sonication as demonstrated.

Transfer the sonicated fragment into a covered glass bottom 35 millimeter dish containing two milliliters of bath solution and mount the dish onto an inverted microscope equipped with a high numerical aperture 60x water immersion objective. Check that the camera port of the microscope is connected to a spectrograph in series with a high sensitivity CCD camera and use a peristaltic pump to perfuse the bath chamber with buffer at a flow rate of 0.5 to one milliliter of buffer per minute. To identify unroofed membrane fragments expressing the ANAP labeled channel, view the sample to locate membranes with fluorescent channels.

Partially engage the spectrometer mask between the camera port on the microscope and the spectrograph. The shadow of the mask will appear on the camera image. Acquire a bright field and fluorescence image of the unroofed membrane to allow selection of the region of interest for analysis.

Bring the tip of the microvolume perfusion system close to the unroofed membrane. To acquire an image of ANAP fluorescence, excite the membrane with a 385 nanometer LED through a 390/18 nanometer band pass excitation filter and a 416 nanometer edge dichroic, collecting the emitted light through a 400 nanometer longpass emission filter. Engage the spectrometer mask and ensure the emitted light is passed through.

Align the region of interest with the slit of the spectrometer mask. Engage the spectrometer gratings. 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.

The images will retain spatial information in the Y dimension. The X dimension will be replaced with wavelength. While perfusing with nucleotide free buffer solution, obtain one or more 0.1 to ten second exposures for downstream correction and normalization of the data collected throughout the rest of the experiment.

Next, apply a range of TNP ATP concentrations in bath solution to establish a concentration response curve, perfusing each solution for at least one minute to ensure that a steady state is reached and obtain an exposure for each concentration. After each concentration, wash out the TNP ATP with nucleotide free bath solution for at least one minute before acquiring a post-treatment exposure image. To perform a patch-clamp fluorometry experiment, first pull patch pipettes from thick walled borosilicate glass capillaries to a resistance of 1.5 to 2.5 megaohms when filled with pipette solution.

Next, transfer a cover slip with transfected cells into glass bottom 35 millimeter dish containing two milliliters of bath solution and mount the dish onto the inverted microscope. Perfuse the bath chamber with bath solution and identify a cell expressing ANAP labeled channels. Apply gentle positive pressure to a patch pipette and place the pipette in a bath chamber.

Press the pipette against the membrane of the cell and apply gentle suction to achieve a gigaohm seal. To excise the patch, rapidly move the pipette holder away from the patched cell. 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.

Then apply TNP ATP and collect image spectra. In these images, a typical unroofed membrane fragment from a HEK293T cell expressing ATP sensitive potassium channels tagged with orange fluorescent protein can be observed. In this spectral image of ANAP tagged ATP sensitive potassium channels in an unroofed membrane from a HEK293T cell exposed to five micromolar TNP ATP, two regions of high intensity can be observed that correspond to the peak emission of ANAP and TNP ATP.

A final spectrum can be obtained by subtracting the averaged background spectrum from the averaged region of interest spectrum. Here, the reduction in peak ANAP fluorescence after multiple exposures can be observed. The peak fluorescence from several exposures in the absence of TNP ATP was fitted to a single exponential decay.

These data were then used to correct photobleaching artifacts. Here, representative spectral images from an unroofed membrane obtained from a cell expressing ANAP tagged ATP sensitive potassium channels in the absence and the presence of TNP ATP are shown. Observing the corrected emission spectra reveals a clear separation between the donor and acceptor fluorescent emission.

In this figure, representative currents and spectra from ANAP tagged ATP sensitive potassium channels from a typical patch-clamp fluorometry experiment are shown. The emission spectra are corrected for background and bleaching as demonstrated for unroofed membranes. Take care with controls to verify that the fluorescent signal is specific to your receptor.

Make sure your profusion is fast, efficient, and complete and keep your exposures as short as possible. This method answers questions about receptor occupancy and how it relates to function. Other FRET techniques used with a similar setup can probe ligand-induced confirmational changes in the receptor.