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.
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withdrawn while the gigaseal is maintained.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
- 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gently withdrawn.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
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.