The Patch-Clamp Method
An improved voltage-clamp technique
Electrophysiology
-Is the study of the electrical properties of biological cells
and tissues.
-It involves measurements of voltage change or electric
current on a wide variety of scales from single ion
channel proteins to whole organs like the heart.
-In neuroscience, it includes measurements of the
electrical activity of neurons, and particularly action
potential activity.
History: Voltage-clamp method
-This technique is used to measure the ion currents through the membranes of excitable
cells while holding the membrane voltage at a set level.
-A basic voltage clamp will iteratively measure the membrane potential, and then change
the membrane potential (voltage) to a desired value by adding the necessary current.
-This process figuratively "clamps" the cell membrane at a desired constant voltage,
allowing the voltage clamp to record what currents are being delivered.
-In 1952, scientists Alan Lloyd Hodgkin and Andrew Huxley used this technique to reveal
how ion channel events affect action potentials using this technique.
-At this point in time, however, the technique could only be applied to large cells as sharp
microelectrodes were needed to penetrate the membrane.
History: Patch-clamp method
-This technique allows the study of single or multiple ion channels in cells to be
done.
-Instead of inserting microelectrodes directly into the cells, a micropipette is
instead used to enclose and create a seal on a “patch” of the cell membrane.
-In the late 1970s, Bert Sakmann and Erwin Neher refined the voltage-clamp
technique to this new technique (at the time) and, for the first time, resolved
single ion channel currents across a membrane patch of a frog skeletal muscle.
History: Giga seal improvement
-The patch-clamp method was itself improved upon by the invention of the giga
seal by Ernst Sakmann in 1980, which massively improved the signal-to-noise
ratio and allowed the recording of even smaller electric currents.
-To obtain this high resistance seal, the micropipette is pressed against a cell
membrane and suction is applied. A portion of the cell membrane is suctioned
into the pipette which, if formed properly, creates a resistance in the 10–100
gigaohms range, subsequently called a "gigaohm seal" or "giga seal".
Patch-clamp technique
-Patch clamp recording uses a glass micropipette, called a patch pipette, as a recording
electrode, and another electrode in the bath around the cell, as a reference ground electrode.
-This small size is used to enclose a membrane surface area or "patch" that often contains just
one or a few ion channel molecules.
-Depending on the experiment, the interior of the pipette can be filled with a solution matching
the ionic composition of the bath solution, as in the case of cell-attached recording, or matching
the cytoplasm, for whole-cell recording.
-The researcher can also change the content or concentration of these solutions by adding ions
or drugs to study the ion channels under different conditions.
Variations: cell-attached patch
-For this method, the pipette is sealed onto the cell membrane to obtain
a gigaseal, while ensuring that the cell membrane remains intact.
-This allows the recording of currents through single, or a few, ion
channels contained in the patch of membrane captured by the pipette.
-By only attaching to the exterior of the cell membrane, there is very
little disturbance of the cell structure.
-
Also, by not disrupting the interior of the cell, any intracellular
mechanisms normally influencing the channel will still be able to
function as they would physiologically
Variations: Inside-out patch
-In the inside-out method, a patch of the membrane is attached to the patch
pipette, detached from the rest of the cell, and the cytosolic surface of the
membrane is exposed to the external media, or bath.
-To achieve this, the pipette is attached to the cell membrane as in the cellattached mode, forming a gigaseal, and is then retracted to break off a patch of
membrane from the rest of the cell.
-One advantage of this method is that the experimenter has access to the
intracellular surface of the membrane via the bath and can change the chemical
composition of what the surface of the membrane is exposed to.
-This is useful when an experimenter wishes to manipulate the environment at
the intracellular surface of single ion channels.
Variations: Whole-cell patch
-Whole-cell recordings involve recording currents through multiple channels
simultaneously, over the membrane of the entire cell.
-The electrode is left in place on the cell, as in cell-attached recordings, but more suction
is applied to rupture the membrane patch, thus providing access from the interior of the
pipette to the intracellular space of the cell.
-The advantage of whole-cell patch clamp recording over sharp microelectrode recording
is that the larger opening at the tip of the patch clamp electrode provides lower resistance
and thus better electrical access to the inside of the cell.
-A disadvantage of this technique is that because the volume of the electrode is larger
than the volume of the cell, the soluble contents of the cell's interior will slowly be
replaced by the contents of the electrode. This is referred to as the electrode "dialyzing"
the cell's contents. After a short while (approx. 10min.), any properties of the cell that
depend on soluble intracellular contents will be altered.
Variations: Perforated patch
-Similar to Whole-cell patch
-This variation of the patch clamp method is very similar to the whole-cell configuration. The
main difference lies in the fact that when the experimenter forms the gigaohm seal, suction is
not used to rupture the patch membrane. Instead, the electrode solution contains small
amounts of an antifungal or antibiotic agent, such as amphothericin-B, nystatin, or gramicidin,
which diffuses into the membrane patch and forms small pores in the membrane, providing
electrical access to the cell interior.
-Advantages of the perforated patch method, relative to whole-cell recordings, include the
properties of the antibiotic pores, that allow equilibration only of small monovalent ions between
the patch pipette and the cytosol, but not of larger molecules that cannot permeate through the
pores.
-Disadvantages are the membrane under the electrode tip is weakened by the perforations
formed by the antibiotic and can rupture. If the patch ruptures, the recording is then in wholecell mode, with antibiotic contaminating the inside of the cell
Variations: Outside-out patch
-places the external rather than intracellular surface of the cell membrane on the outside of the
patch of membrane, in relation to the patch electrode
-The formation of an outside-out patch begins with a whole-cell recording configuration. After
the whole-cell configuration is formed, the electrode is slowly withdrawn from the cell, allowing
a bulb of membrane to bleb out from the cell. When the electrode is pulled far enough away,
this bleb will detach from the cell and reform as a convex membrane on the end of the
electrode, with the original outside of the membrane facing outward from the electrode.
-Outside-out patching gives the experimenter the opportunity to examine the properties of an
ion channel when it is isolated from the cell and exposed successively to different solutions on
the extracellular surface of the membrane. The experimenter can perfuse the same patch with
a variety of solutions in a relatively short amount of time
-On the other hand, it is more difficult to accomplish. The longer formation process involves
more steps that could fail and results in a lower frequency of usable patches.
Variations: Loose patch
-Loose patch clamp is different from the other techniques discussed here in that it employs a loose
seal (low electrical resistance) rather than the tight gigaseal used in the conventional technique.
-To achieve a loose patch clamp on a cell membrane, the pipette is moved slowly towards the cell,
until the electrical resistance of the contact between the cell and the pipette increases to a few times
greater resistance than that of the electrode alone.
-A significant advantage of the loose seal is that the pipette that is used can be repeatedly removed
from the membrane after recording, and the membrane will remain intact. This allows repeated
measurements in a variety of locations on the same cell without destroying the integrity of the
membrane.
-This flexibility has been especially useful to researchers for studying muscle cells as they contract
under real physiological conditions, obtaining recordings quickly, and doing so without resorting to
drastic measures to stop the muscle fibers from contracting.
-A major disadvantage is that the resistance between the pipette and the membrane is greatly
reduced, allowing current to leak through the seal, and significantly reducing the resolution of small
currents.
A patch-clamp study of bovine chromaffin cells and of their
sensitivity to acetylcholine. Fenwick E.M., Marty A., Neher E.
-When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached
configuration') current wave forms due to intracellular action potentials could be
observed. When acetylcholine was present in the pipette solution, acetylcholineinduced single channel currents were evident in the patch recording.
-After rupture of the patch of membrane, the pipette--cell seal remained stable
(whole-cell recording). Chromaffin cells were found to have a resting potential of -50
to -80 mV, and a high input resistance.
Tl;dr: Patch-clamp method
-No literal clamps involved;
-A modification of the voltage-clamp method where
one uses a micropipette to create a giga seal around a
single or several ion channels in a cell membrane.
-4 different variations used, each measure the amount
of ion flow going either into or out of the channel(s), but
each allows the separate manipulation of either
internal and external solutions, or both.
Questions?