Comparative characteristics of bacterial protein toxins used as tools for cell permeabilisation
12.3. Methods
12.3.3. Open-Cell Cell- Cell-Attached Patch-Clamp
Configuration
4. The volume of the bath solution is set to 100–200 µl by using a pre-designed perfusion chamber or a perspex insert fitting into a 35-mm Petri dish. Given the number of β-cells on each dish of around 100 and the mean osmotically active volume of a β-cell of 0.8 pl (at 300 mosmol/l) we get the total osmot-ically active volume of the β-cells on the dish of 80 nl. This is less than 0.1% of the total volume of the bath solution, which is sufficient for an efficient “concentration clamp”.
5. In the cell-attached mode, at the pipette potential Vp = 0 mV (Vm ≈ −60 to −70 mV), single openings of KATP channels can be observed (Fig. 12.2). The extracellular solution is then replaced by the intracellular solution containing 100 µM MgATP (see Note 8), and Vm is set to −60 mV (Vp = 60 mV) to restore the gradient of electrochemical potential cancelled by symmetrical [K+], [Na+] and [Cl−].
6. (Optional) Propidium iodide (PI; 3.3 mg/l) can be added into the bath solution either at this stage, or after the addition of
Fig. 12.2.Representative recording of the KATP channel activity in open-cell configuration. Addition of ligand (MgATP) is indicated by horizontal bars. Time points of addition of α-toxin and acquirement of PI-staining by the cell nucleus are indicated with arrows. The time required for diffusion of 100 µM MgATP into the cell is marked as tlag. Parts of the trace marked as AA′ and BB′ are displayed on an expanded timescale in the insets below.
the toxin, to monitor the permeabilisation. We recommend adding the dye using an automatic pipette or injection sys-tem. Please note that frequent exposure to PI may result in non-specific staining of glass or plastic surfaces.
7. The toxin is then introduced into the solution. The perfusion is stopped, and 5–10 µl of the toxin is added into the bath solution with a 10 µl automatic pipette (Gilson), as a drop-let, close to the patch electrode. The addition introduces a short noise transient into the recording (see Fig. 12.2);
however it very rarely damages the seal. For other methods of adding toxin into the solution see Note 1 and Note 4.
8. The toxin is left to diffuse, bind to the cell membrane and oligomerise, and within 1–2 min the first signs of permeabi-lisation can be observed. The activity of KATP channels may decrease, possibly due to the complete cancellation of [Na+] and [K+] gradients, which would reduce the utilisation of ATP by the sodium pump (9), whilst the baseline shifts in a more positive direction and becomes less “noisy” (see Note 6).
9. The ensuing increase of PI (decrease of FDA) fluorescence and gradual restoration and increase of the KATP activity, due to the ATP wash-out, confirm the fact of permeabi-lisation (Fig. 12.2). If permeabipermeabi-lisation-related effects are not observed, one more 5 µl droplet of the toxin should be added to the solution, etc., until the cell is clearly permea-bilised. Likewise, more of the toxin should be added if the onset of the permeabilisation is too slow.
10. 3–10 min after the permeabilisation, when the KATP activity has reached a stable level, the perfusion of the bath solu-tion is re-started and the channel activity can be recorded in response to various agents. See however Note 9.
The fluorescent dyes propidium iodide (PI) and fluorescein diacetate (FDA) are used to visualise the permeabilisation. The bright-field dye trypan blue is used to assay the toxin activity.
Counting the cells by eye is technically easier in the bright field;
therefore non-fluorescent staining has an advantage.
1. PI, a fluorescent DNA intercalant (λex = 535 nm, λem = 615 nm, MW = 668; compare with 507 of ATP), is membrane-imper-meant but does permeate through α-toxin pores. Upon the permeabilisation of the plasma membrane, the cell acquires nuclear red fluorescence, while the cell cytoplasm remains PI-negative. PI is dissolved in the intracellular solution to the working concentration of 3.3 mg/l either before application of the toxin or simultaneously or shortly after.
2. FDA is membrane permeant but in the cytoplasm it gets hydrolysed into non-permeant fluorescein (λex = 494 nm, 12.3.4. Visualisation of
Cell Permeabilisation
lem = 517 nm, MW = 332). The working concentration for FDA is 0.5 mg/l. Intensive leakage of fluorescein through the toxin pores can also be taken as an indication of permeabi-lisation. One should bear in mind however, that fluorescein does (slightly) leak through the intact membrane and has a high rate of photobleaching.
3. Both PI and FDA have broad emission and excitation spec-tra, which makes it possible to visualise both of them simul-taneously, using the same filter set. Combining a 470 ± 20 nm excitation filter, a 520 ± 20 nm emission filter and a 510 nm dichroic mirror allows one to observe bright green fluorescein emission and weaker but distinct emission of PI in the orange range of the spectrum.
1. The main driving force of the mass-exchange through the permeabilised membrane, in the open-cell experiments, is likely to be the gradient of chemical potential rather than the hydrostatic pressure.
2. A short lag-period (<60 s for experiments using α-toxin but longer in the case of SLO, see Note 10) is always observed between addition of the ligand (MgATP) and changes in the channel activity (tlag in Fig. 12.2). The over-extension of the lag-period reflects insufficient permeabilisation, the lack of the lag-period means the patch is excised from the cell surface.
3. In contrast to what is observed in the inside-out patches (10), the run-down of the KATP channel is quite small, in the open-cell configuration. The control (ligand-free) solution can therefore be applied only at the start and at the end of the experiment.
4. The duration of one open-cell experiment, which can be up to 1 h depends on the number of conditions tested. This sets limits for the acquisition rate. The acquisition software should be able to support long segments of continuous acquisition, with or without repeated stimulation, with on-line input of labels and comments. pClamp (Molecular Devices) is par-ticularly recommended, Pulse (HEKA) requires insertion of a continuous segment and pre-loading of the solution data-base. Monitoring the recording on two timescales – “fast” for single-channel events and “slow” for the time-course of the channel activity – is a good idea.
5. Analysis of the continuous data assumes idealising the trace by counting the channel opening and closing events and fur-ther deriving the channel characteristics (Po, rate constants, etc.), in exactly the same way as is done for recordings from the inside-out configuration. The data corresponding to the diffusion of the ligand (tlag in Fig. 12.2) should be excluded from analysis.
12.3.5. Recording and