Jonathan D. Lippiat
11.4. Notes
are typically obtained. A sample recording under these con-ditions is shown in Fig. 11.3.
1. Although filamented capillaries can be used, the lack of a fila-ment in the capillary glass gives better “tipping” partition-ing between amphotericin-containpartition-ing and amphotericin-free solutions (see Subheading 11.3.3.1).
2. The pipette solution is designed to closely match the intra-cellular concentrations of K+, Na+, and Cl−, which permeate amphotericin B pores. Whilst SO42− is used here, other suit-able impermesuit-able counteranions, such as aspartate, may also be used. The gradual exchange of ions between cytosol and patch pipette to reach the Donnan equilibrium results in a voltage drift over time.
3. Cells can be plated onto glass cover slips if preferred and coat-ing agents (e.g. poly-D-lysine) used if poorly adherent.
4. Do not attempt to mix with the pipette tip as the amphotericin B dissolved in DMSO will stick to the plastic tip. Flick the microtube to gather the contents to the bottom, dispense the DMSO along the side of the tube, and then sonicate.
5. The use of a glass container is recommended as the ampho-tericin–DMSO sticks to plastic.
6. This will appear as an apparently insoluble yellow “slug” at the bottom of the flask. Dissolve the “slug” by repeated pipetting with a 1 ml pipette (e.g. Gilson P1000).
7. The foil prevents destruction of the antibiotic by light.
8. Patch pipettes should be prepared as if they were to be used in standard whole-cell recording with relatively low resistances to reduce the membrane charging time constant and series-resistance errors. It is critical that patch pipettes are clean and polished to ensure optimal seal formation in as short a time possible, and it is worthwhile optimising the cell preparation and patch pipette fabrication.
9. The presence of amphotericin B in the pipette solution can prevent seal formation, and likewise any amphotericin B that leaks from the patch pipette near the cell can damage the membrane. By “tipping” the pipette tip with antibiotic-free solution, optimal seal formation should take place before the amphotericin reaches the tip where it invades the mem-brane. How much antibiotic-free solution should be loaded into the patch pipette tip (i.e. how long the patch pipette should
be held in the drop of antibiotic-free solution) requires opti-mising and depends on how quickly gigaohm seals are formed.
The more a patch pipette is “tipped”, the longer it takes for the amphotericin to arrive at the aperture and perforate the membrane. The antibiotic-free solution should not go beyond 0.5 mm from the aperture.
10. Amphotericin can readily come out of the solution or stick to the sides of the container. This is remedied by mixing by swirling the container prior to use.
11. This approach is preferred to using a conventional patch pipette filling syringe containing the amphotericin pipette solution as it enables the amphotericin to be mixed immedi-ately prior to use, and avoids exposure to light, plastic, and filtering.
12. With the use of unfilamented capillary glass, there is a ten-dency for air bubbles to form at the interface between the two solutions. Dispense the amphotericin solution as close to the antibiotic-free “tip” as possible, and flick the pipette gently to remove bubbles from the taper.
13. This is usually done via tubing that is connected to a 1-ml syringe. Positive pressure is applied by pushing the plunger to the equivalent of 0.1 ml. The purpose is to maintain an outward flow of clean solution that prevents contamination from the bath solution and tissue. However, the pressure needs to be kept to a minimum so that the amphotericin-containing solution is not expelled over the tissue prior to seal formation.
14. The solutions described here give a junction potential of approximately +10 mV. The calculated or measured junc-tion potential can be accounted for, prior to seal formajunc-tion.
This can be done automatically if the amplifier is computer driven, or manually by “zeroing” to the junction potential using the DC-offset with the amplifier in current clamp mode.
15. Contact between HEK293 cells results in electrical coupling via gap junctions, which will prevent single cell recording.
Membrane currents from the additional cells will be con-ducted via gap junctions. Evidence for this can be observed from the whole-cell capacitance where additional compo-nents with a longer time constant (and therefore through higher access resistance) are observed.
16. Suction can be applied using a 1 ml syringe (see Note 13).
Sealing can be assisted using negative holding potentials, but determining the optimum sealing procedure using the cells, patch pipettes, and solutions without amphotericin is strongly recommended.
17. The time-course of the capacitance transient follows the exponential function I = I0e−t/t, where I0 is the instantane-ous current amplitude and is the time constant. I0 follows Ohms Law, I0 = V/Ra, where V is the amplitude of the test pulse and Ra is the access resistance (see Fig. 11.2). The time constant equates to RaCm, where Cm is the membrane capacitance. As Ra decreases with increased perforation, I0 increases and decreases.
I thank Dr. Peter Proks (Oxford) and Dr. John Linley (Leeds) for helpful discussion regarding this technique, and the Welcome Trust and BBSRC for financial support.
Acknowledgments
References
1. Horn R. and Marty A. (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen.
Physiol. 2, 145–59.
2. Rae J., Cooper K., Gates P., and Watsky M.
(1991) Low access resistance perforated patch recordings using amphotericin B.
J. Neurosci. Methods 37, 15–26.
3. Horn R. (1991) Diffusion of nystatin in plasma membrane is inhibited by a glass-membrane seal. Biophys. J. 60, 329–33.
4. Toye A.A., Lippiat J.D., Proks P., Shi-momura K., Bentley L., et al. (2005) A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice. Diabetologia 48, 675–86.
5. Tsuboi T., Lippiat J.D., Ashcroft F.M., and Rutter GA. (2004) ATP-dependent interac-tion of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by flu-orescence resonance energy transfer. Proc.
Natl Acad. Sci. USA 101, 76–81.