Bradykinin Research
Perforated Patch Clamp
The Patch Clamp technique, devised in the 1970s, is based on a simple idea; a small diameter glass pipette is used to make a seal with a small patch of neuronal cell membrane. Suction is then applied via the pipette to form a tight seal, which does not allow any ionic flow between the pipette and the membrane. This ensures that any ions flowing through membrane ion channels can only flow between the cell and the pipette. This isolated current can be recorded using sensitive amplification equipment attached to the pipette. This method, like voltage clamping, also allows control of the membrane potential, in order to allow characterization of the voltage dependent nature of membrane currents.
This technique has been further developed to allow more specific uses. Perforated Patch Clamping allows the measurement of whole-cell currents, single-channel currents and transmembrane voltages much less invasively than standard patch-clamp or microelectrode approaches.
The technique utilizes either nystatin or amphotericin B (polyene antibiotics) to gain electrical access to the cell's interior by forming channels in the membrane. The channels produced are permeable to monovalent cations and Chlorine ions (Cl-) (the monovalent ions being more permeant) but do not allow passage of multivalent ions, eg. Mg2+ or Ca2+.
Because of their limited water solubility, amphotericin and nystatin stock solutions of 30 mg/ml are prepared in DMSO; they are prepared just before use due to their loss of activity on prolonged storage. Stock solution can be added directly to the desired pipette filling solution to a final concentration of 120 - 350 µg/ml. With both antibiotics, the solution is yellow in colour and slightly cloudy but after filtration the final solution is clear.
Both compounds appear to interfere with seal formation; the success rate of fully sealing a cell is far lower that with an antibiotic-free solution is used. As such, the pipette tip can be filled with antibiotic-free solution and the rest with the antibiotic-saturated solution. The amount of antibiotic-free solution must be adjusted to allow for the varying time needed to form a seal, which is cell dependent.
The perforated-patch technique has several advantages over conventional whole-cell patch-clamp approaches:
- The channels formed are impermeable to molecules as large as (or larger than) glucose. Therefore, whole-cell recordings can be done without dialyzing important substances from the cell's cytoplasm – this ensures the continued operation of important cell signaling and channel regulation intracellular (i.c.) cascades and mechanisms.
- i.c. multivalent ions are not affected by the pipette-filling solution. Therefore, i.c. Ca2+, for example, can be measured by optical techniques simultaneously with recording whole-cell currents.
- The perforation technique is less damaging to cells than intracellular microelectrodes or standard whole-cell patch clamping.
- Seals, once obtained, are easily maintained.
- Whole-cell capacitance is usually easier to compensate with the perforated technique.
The perforated-patch technique does have some disadvantages as well:
- It does not allow dialysis of the cell's cytoplasm and hence its contents cannot be substituted with compounds other than the small ions included in the filling solution. Therefore, the effects of such compounds on intercellular mechanisms cannot be studied.
- The pipette must contain an impermeant anion and a Cl- concentration that match the cell interior. Failure to do so results in a Cl- flux either into or out of the cell (depending on the direction of the mismatch). In addition the cells that are voltage clamped by the perforated-patch technique are very susceptible to volume changes caused by external perturbations.
- The perforation process requires considerably longer time for achieving access into the cell interior. When a rapid measurement from a large number of cells is important, standard whole-cell patch-clamp approaches would be more productive.
- While the relative Na+, K+ and Cl- permeabilities of nystatin and amphotericin channels are known, the permeation by other anions and cations, particularly multivalent ions that are generally considered impermeant, needs further study.
