What is patch clamping?
The Patch Clamp Technique
Since its introduction by Professors Bert Sakmann and Erwin Neher in the mid 1970’s, patch clamp has been the classic method for studying ion channel function. It allows direct measurement of single channel currents as well as of the total current across the entire cell membrane. A glass micro-pipette, containing an electrolyte and an electrode, is pressed against the cell membrane, and a piece of membrane (the ‘patch’) is positioned within the pipette orifice. A tight seal of gigaohm electrical resistance (a ‘gigaseal’) is formed between the pipette rim and the cell membrane. If the patch contains ion channels then movement of ions through these channels is measured as tiny (picoampere) currents. The leak current across the seal is insignificant due to the high resistance of the gigaseal.
Specifically, five configurations may be employed:
- Cell-attached (on-cell): the pipette makes a gigaseal with the intact cell, allowing measurements of single-channel currents.
- Inside-out: cell-attached configuration is achieved and the pipette is withdrawn while the gigaseal is maintained, rupturing the cell. The inside (cytoplasmic) side of the membrane faces the bath fluid. This configuration is used for single-channel recordings with the ability to change the ‘intracellular’ solution.
- Whole-cell: the cell-attached configuration is achieved and vigorous suction is applied to the pipette, causing the patch to break. The cytoplasm and the pipette solution are subsequently in direct contact. After a short time, diffusion of cytoplasmic constituents (molecules and cell organelles) leads to identical (unphysiological) chemical composition of the fluids in the cell and in the pipette. The activity of all membrane ion channels is measured in this configuration.
- Outside-out: the whole-cell configuration is achieved and the pipette is gently withdrawn. This causes the membrane to break outside the sealing zone. The membrane fragments subsequently flip over, reseal, and constitute an inverted membrane patch exposing the extracellular side to the bath fluid. This configuration is used for single-channel recordings.
- Perforated whole-cell: the cell-attached configuration is achieved with pore-forming compounds (e.g. amphotericin B, nystatin) in the pipette solution. This causes perforation of the patch allowing small molecules and ions, but not larger compounds, to cross the patch. Consequently, larger molecules and cell organelles remain within the cell. The sum of all ion channel currents is measured, as with conventional whole-cell patch clamp.
Patch clamp has proven particularly powerful In combination with molecular biological techniques: specific ion channels may be expressed in cultured cell lines allowing a thorough characterization of their biophysical and pharmacological properties by patch clamp.
Patch clamp technologies for high-thoroughput screening
Unfortunately patch clamp is a low throughput technique. It is time-consuming and demands a skilled operator. The QPatch family and the Qube systems enable automated parallel ion channel screening with throughputs of hundreds or thousands of data points per day, enabling faster, and more accurate drug discovery.