Adi Raveh, Inbal Riven, and Eitan Reuveny
16.1. Introduction
Chapter 16
The Use of FRET Microscopy to Elucidate Steady State
and on the distance between the donor and the acceptor mol-ecules. In general these two variables will determine the efficiency of transfer. In more practical situations, where the donor and the acceptor are free to rotate around a certain axis, the distance between the donor and the acceptor is the main determinant influencing the efficiency of resonance transfer (1). The depend-ence of the transfer efficiency on distance is very high, e.g., raised to the sixth power. Thus minute change in the distance between the donor and the acceptor chromophores is translated to a big change in FRET efficiency. For this reason the FRET techniques has been used as an inter- and/or intramolecular ruler, operating in the range between 1 and 10 nm (2). The FRET technique also has its limitations and various considerations have to be taken once quantitation is in mind (3–5). One of the ways to estimate the FRET efficiency between two fluorophores is to rely on the amount of donor emission, the closer the fluorophores the larger the dequenching (reduction in emitted photons) of the donor (5). Once one has the mean of eliminating the acceptor molecule, the ratio of donor emission in the presence of the acceptor to a condition where the acceptor is nonfunctional, e.g., bleached, will determine the FRET efficiency (6, 7).
There are many ways to detect the emitted fluorescence; the most conventional one is to use a specific emission filter to allow only photons within a certain wavelength to pass through, and to be detected by a photomultiplier or by an avalanche photodi-ode. This arrangement has a high temporal resolution, but lacks the spectral information. This may be important when informa-tion is required regarding the spectral characteristics of the emitted photons. In our studies we used a spectrometer and a back illumi-nated CCD array to capture spectral information from our sample.
The signal intensities at the expected emission peak of the donor were measured before and immediately after photo-bleaching of the acceptor, and then were used to calculate the FRET efficiency (E).
Distances between the donor and the acceptor chromophores were then calculated based on E = R06 /(R06+R6), where R0 (Förster dis-tance) is the distance when E = 0.5. R0=(9.7×103 J fD n– 4k 2)16, 50.4 Å for CFP/YFP pair (8). In our previous studies (9, 10) we utilized the advantages of FRET microscopy to study the rearrangement of G protein-coupled potassium channels (GIRK/Kir3.x) upon their gating and their mode of interaction with G proteins. GIRKs are inwardly rectifying K+ channels that generate slow, inhibitory post-synaptic potentials upon activation by Pertussis-toxin-sensitive G protein-coupled receptors (GPCRs) (11).
GIRK activation requires the binding of Gβγ to the channel.
Upon neurotransmitter release from the presynaptic cleft and GPCR stimulation at the postsynaptic site, GTP is exchanged for GDP on the Gα subunit of the G protein. This, in turn, leads to the dissociation of a Gβγ subunit from the Gα subunit, to freely
interact with the channel’s Gβγ binding domains. The binding of Gβγ induces a conformational change of the channel promoting the opening of the permeation pore to allow the selective flux of K+ ions through it. The flow of ions is mainly in the outward direction which then hyperpolarizes the postsynaptic membrane, to produce a reduction in excitation.
There is no full length three-dimensional structure of the GIRK channel. Nevertheless, the structure of the conserved regions (regions that are shared by other inwardly rectifying chan-nels from bacteria to vertebrates, of both the N- and C-terminal cytosolic domains) have been solved by X-ray crystallography (12–14). These structures of the cytosolic domains in conjunc-tion with solved structures of the membrane core of other similar transmembrane potassium selective channels, KcsA, MethK and KirBac, provide a general static view of the channels presumably in the closed and open conformations (12, 15–17); for a review see ref. 18. Despite the knowledge accumulated as described above, we still do not have a detailed mechanistic understand-ing of the conformational changes that allow channel gatunderstand-ing of inwardly rectifying potassium channels, in particular those that are activated by intracellular modulators, like G proteins.
We took advantage of the known stoichiometry of the GIRK channels and the fact that one of the subunits, GIRK1, is unable to form homotetramers. In our work we used the cardiac version of GIRK channels, mainly heterotetramers of GIRK1/GIRK4 in a 1:1 ratio, thought to be arranged in a fourfold symmetry (19–21). This turns out to be of a great advantage, since FRET pairing of known stoichiometry allows quantitative interpretation of the FRET signals. We chose to tag both the cytoplasmic N- and C-termini of these subunits by the fluorescent FRET pair CFP (the donor) and YFP (the acceptor) (8, 22). CFP and YFP are a good FRET pair, having a relatively large overlap between CFP emission spectrum and YFP absorption spectrum, which is crucial for effi-cient energy transfer. In addition, their absorption spectra enable selective excitation of the donor with minimal or complete absence of acceptor excitation. It is very important to test the functionality of the tagged channel subunits. All fused constructs were thus imaged to validate proper translocation to the plasma membrane, and tested electro-physiologically for their functionality (10).
Measuring FRET between tagged neighboring subunits in each combination (two termini for each of the subunits) allowed us to measure the distances between the neighboring subunits and to translate each distance to a point in X, Y, Z space.
Two kinds of FRET measurements were performed, to elu-cidate structural changes associated with channel gating and to understand the relationship between the channel and the G pro-tein subunits, at rest and during activation. The former measure-ments were conducted for both closed and open channels (by both
over-expression of Gβγ or following activation of the A1 adenosine receptor). Comparison of the space representation of the channel’s cytosolic ends in its closed, vs. open states enabled us to create a model that predicts the general vectorial change associated with channel gating. The relationships between the GIRK channel and the G proteins subunits were studied by tagging the channel subunit with the donor fluorophore and the appropriate G protein subunits with the acceptor. In both types of measurements, the technicality of the experiment is similar and thus will be discussed without refer-ring to any particular set of experiments independently. In some cases, we will describe the detailed calculation methodology which is specific for the specific type of experiment presented.
To increase the confidence that changes seen in FRET effi-ciency are indeed due to changes in the distance between the fluorophores, one has to verify that the relative mobility of the donor and/or the acceptor is not changing during the confor-mational transitions. We thus also describe the methodologies used to record the acceptor fluorescence anisotropy. Fluorescent anisotropy, r, is a measure of the extent of polarization of the sample. Upon excitation with polarized light, the emission from many samples is polarized. However, rotational diffusion of the fluorophores can lead to depolarization of the emission and thus to a reduced excitation of the neighboring acceptor (1). In our experiments, after correcting for the microscope polarization characteristics (see below), the apparent acceptor anisotropy values were similar for both unstimulated and stimulated cells, indicating that although the acceptor is attached to the channel, fluorophores’ free mobility is not affected following channel gating. Hence, constraints on dipole orientation are unlikely to substantially affect the FRET efficiency or account for FRET effi-ciency changes detected upon channel activation.
In most cases when intracellular fluorescent tags are utilized, they are encoded genetically as fusion to the protein of interest. This approach introduces a complication when used on membrane pro-teins, or any proteins that have a well-defined subcellular distribution.
To specifically image those proteins in their target location, means of spatial control of either the excitation or the emission is desired.
In the case of the GIRK channels the spatial control becomes very important, mainly due to the fact that a substantial amount of the mature fluorescently tagged channels are already present in many intracellular compartments. Therefore, to specifically monitor chan-nel residing exclusively in the plasma membrane, we used total inter-nal fluorescence (TIRF) (23). In TIRF microscopy, on reaching the critical angle (and beyond), the excitation beam totally changes its direction and is reflected from the cover glass. This process is mainly due to the difference of the refraction index of the cover glass and the water (for more reading see refs. 23 and 24). Once the excita-tion beam is reflected from the glass, it generates an electromagnetic standing wave, the evanescent wave (perpendicular to the cover
glass), that has the exact spectral characteristics of the excitation light. The power of this evanescent wave drops exponentially within 100–200 nm, depending on the excitation angle and wavelength of the excitation beam. This useful characteristic of the evanescent wave allows the selective excitation of objects very close to the cover glass, such as the plasma membrane of cells.
Using the described methods we were able to monitor the conformational rearrangements of the GIRK channels during gating and the intricate relationship of this channel with the G proteins, both at rest and during activation (9, 10).
Following is a detailed description of the methodology used to obtain these results as well as points that require special attention.
1. Ø24 mm number 0 cover slips (see Note 1).
2. 1-M NaOH (keep safety precautions and work in chemical hood).
3. 0.01% Poly-l-lysine. Store at 4°C.
4. Borate buffer solution: Boric acid (3.1 g/l), Borax (4.75 g/l).
Store at 4°C.
5. Sterile double-distilled water (DDW).
6. 0.22-µm syringe filter, syringe.
7. 6-well plates.
8. Aluminum foil.
9. Autoclaved glass pipettes and 2-, 5-, and 10-sterile plastic pipettes.
1. Human embryonic kidney (HEK 293) cells.
2. T-25 Tissue culture flasks.
3. 0.25% Trypsin–EDTA.
4. Dulbecco’s Modified Eagle’s Medium (DMEM), HEPES modification, without L-Glutamine.
5. DMEM/F-12 (HAM) 1:1 without L-Glutamine.
6. L-Glutamine solution, 200 mM.
7. Pen-Strep solution (penicillin 10,000 units/ml; Streptomy-cin 10 mg/ml).
8. Fetal calf serum (FCS).
9. 15-ml conical centrifuge tubes, 1.5-ml eppendorf tubes.
10. 35-mm tissue culture dishes.
11. Autoclaved glass pipettes and 2-, 5-, and 10-sterile plastic pipettes.