The Patch Clamp Technique

Since its introduction by Professors Bert Sakmann and Erwin Neher in the mid 70'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. Due to the high resistance of the gigaseal, the leak current across the seal is insignificant.

patch clamp

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  • Inside-out: upon cell-attached configuration the pipette is withdrawn while the gigaseal is maintained. The inside (cytoplasmic) side of the membrane is facing the bath fluid. This configuration is used for single-channel recordings with the ability to change the "intracellular" solution.
  • Whole-cell: upon cell-attached configuration 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. In this configuration the activity of all membrane ion channels is measured.
  • Outside-out: from whole-cell configuration the pipette is gently withdrawn.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: 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 are remaining within the cell. As with conventional whole-cell patch clamp the sum of all ion channel currents is measured.

In combination with molecular biological techniques patch clamp has proven particularly powerful: 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 throughput screening
Unfortunately patch clamp is a low throughput technique.It is time-consuming and demands the presence of a skilled operator. The QPatch family and the Qube systems consists of automated parallel ion channel screening technology systems, with throughputs of hundreds or thousands of data points per day, enabling faster, and more accurate drug discovery.