The patch clamp technique allows researchers to study single or multiple ion channels in cells. It involves pressing a glass pipette against a cell to form a high resistance seal, isolating a patch of membrane. This enables recording of tiny ion channel currents. Variations include cell-attached for intact cells, whole-cell to access the interior, and outside-out to study channels in isolation. The technique was developed in the 1970s-80s and earned Neher and Sakmann the Nobel Prize, as it proved ion channels mediate processes like action potentials.

1. PATCH CLAMP
Presented by:
Syed Kashif
Department of Pharmacology
AACP

2. Erwin Neher
Bert Sakmann
Germany
(1991 Nobel
Laureates)

3. CONTENTS
1.Definition
2.Principle
3.Set up
4.Recording
5.Variations
6.Applications

4. Definition
• The patch clamp technique is a laboratory technique in
electrophysiology that allows the study of single or multiple
ion channels in cells.
• The technique can be applied to a wide variety of cells, but is
especially useful in the study of excitable cells such as
neurons, cardiomyocytes, muscle fibers and pancreatic beta
cells.
• It can also be applied to the study of bacterial ion channels in
specially prepared giant spheroplasts.
• The patch clamp technique is a refinement of the voltage
clamp.
• Erwin Neher and Bert Sakmann developed the patch clamp in
the late 1970s and early 1980s. This discovery made it
possible to record the currents of single ion channels for the
first time, proving their involvement in fundamental cell
processes such as action potential conduction. Neher and
Sakmann received the Nobel Prize in Physiology or Medicine

6. Principle
 The principle of the method is to isolate a patch of membrane
electrically from the external solution and to record current
flowing into the patch
 This is achieved by pressing a fire-polished glass pipette, which has
been filled with a suitable electrolyte solution, against the surface
of a cell and applying light suction

7. The patch clamp circuit
Patch of cell membrane with ion channel
FBR
_
+
Amplifier
Technical
The high gain operational amplifier is
connected in the circuit so that the current
flowing through the ion channel is measured
as a voltage drop across the feedback resistor
(FBR). The FBR has a resistance of 50 G
allowing very small currents (10-12 A)
to be measured.

8. Set up
 Patch clamp recording uses a
glass micropipette called a patch pipette as a
recordingelectrode, and another electrode in the
bath around the cell, as a reference ground
electrode. Depending on what the researcher is
trying to measure, the diameter of the pipette tip
used may vary, but it is usually in
the micrometer range. This small size is used to
enclose a membrane surface area or "patch" that
often contains just one or a few ion channel
molecules

9.  In some experiments, the micropipette tip is
heated in a microforge to produce a smooth
surface that assists in forming a high
resistance seal with the cell membrane. 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, creating an omega-
shaped area of membrane which, if formed
properly, creates a resistance in the 10–
100 gigaohmsrange, called a "giga ohm seal" or
"gigaseal".[3] The high resistance of this seal
makes it possible to isolate electronically the
currents measured across the membrane patch
with little competing noise, as well as providing
some mechanical stability to the recording

10.  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.

13. Variations in patch clamp
 1)Cell-attached or on-cell patch:
 The electrode is sealed to the patch of
membrane, and the cell remains intact.
 This allows for the recording of currents
through single ion channels in that patch of
membrane, without disrupting the interior of
the cell.
 For ligand-gated ion channels or channels
that are modulated by metabotropic
receptors, the neurotransmitter or drug being
studied is usually included in the pipette
solution, where it can contact what had been
the external surface of the membrane.

15.  2)Whole-cell recording or whole-cell patch:
 Whole-cell recordings, in contrast, involve recording
currents through multiple channels at once, over the
membrane of the entire cell.
 The electrode is left in place on the cell, but more
suction is applied to rupture the membrane patch, thus
providing access to the intracellular space of the cell.
 Advantage: 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.
 Disadvantage:Is that the volume of the electrode is larger
than the cell, so 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.

17.  3)Outside-out patch:
 After the whole-cell patch is formed, the
electrode can be 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
(like a ball open at the electrode tip). with the
original outside of the membrane facing outward
from the electrode.
 Single channel recordings are possible in this
conformation if the bleb of membrane is small
enough.

18.  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 to
different solutions on the extracellular
surface of the membrane.
 Advantage:The experimenter can perfuse
the same patch with different solutions.
 Disadvantage:It is more difficult to
accomplish, as more steps are involved
in the patching process.

20.  4)Perforated patch:In this variation of
whole-cell recording, the experimenter
forms the gigohm seal, but does not
use suction 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.
 As the antibiotic molecules diffuse into
the membrane patch, they form small
perforations in the membrane,
providing electrical access to the cell
interior.

22.  5)Loose patch:
 Loose patch clamp is different in that it
employs a loose seal rather than the tight
gigaseal used in the conventional technique.
 Advantages: Is that the pipette that is used can
be repeatedly removed from the membrane
after recording, and the membrane will
remain intact.
 This allows for repeated measurements in a
variety of locations on the same cell without
destroying the integrity of the membrane.
 Disadvantage: Is that the resistance between
the pipette and the membrane is greatly
reduced, allowing current to leak through the
seal.

24. Procedure
 Acute brain slices / cultured cells / enzymatically isolated cells should
be superfused in ACSF / extracellular solution and continuously gassed
with carbogen (5% CO2/95% O2) for at least 2 h at room temperature
before recording.
 Pull recording microelectrodes to an input resistance of 5–8 MΩ.
▲ CRITICAL STEP
 Set the bath application system to run at 1–2 ml per minute. Place the
slice/cells in.
 Fill the recording microelectrode with electrode solution.
 If documenting cellular morphology post hoc is desired, include the
intracellular dye filling of choice in the micropipette solution. Available
dyes include Lucifer yellow, Cell Tracker, biocytin, Alexa biocytin,
neurobiotin, etc.
 Place the microelectrode in the pipette holder. Apply positive pressure
using a 10-ml syringe by displacing the plunger about 1 ml.
▲ CRITICAL STEP
 Set the amplifier to voltage clamp and apply a test pulse of 5–10 msec
and 20 mV amplitude. Slowly approach the area of interest until there is
an obvious change in the test pulse amplitude.

25.  Once an obvious and steady change in
microelectrode resistance is obtained, release
the positive pressure rapidly.
▲ CRITICAL STEP
 Obtain a GΩ seal spontaneously. If not, briefly
apply light suction by mouth until the resistance
reaches at least 1 GΩ.
 Once a GΩ seal has been formed, proceed to
obtain the desired patch-clamp configuration.
◦ Cell-attached configuration: Upon acquiring a GΩ
seal one can proceed with the experiment. In this
configuration, the microelectrode solution should
resemble extracellular medium.
◦ Inside-out configuration: After obtaining a GΩ seal,
slowly pull the pipette away from the cell. Eventually,
a small piece of cell membrane will be detached from
the cell surface without losing the GΩ seal. The
microelectrode solution should resemble extracellular
medium in this configuration also.

26. ◦ Whole-cell configuration:
 In this configuration, the microelectrode solution should
approximate intracellular ionic composition.
 To record in whole-cell mode, change the voltage clamp to
a negative voltage close to the cell resting potential (–60
mV for radial glial cells) and correct for fast capacitance.
Apply continuous light suction by mouth until the
membrane breaks as evidenced by a change in the
capacitance and the test pulse current. ▲ CRITICAL STEP
 If doing perforated patch recordings, front-fill the
microelectrode with electrode solution without antibiotic,
back-fill with electrode solution containing antibiotic. After a
GΩ seal is obtained, simply set the voltage clamp near the
resting potential and wait for the resistance to slowly
decrease and stabilize.
◦ Outside-out configuration: After obtaining a whole-
cell recording, very slowly withdraw the pipette
until resistance increases greatly, indicating
formation of an excised membrane ‘bleb.’
 Analyze recordings. Most acquisition software
comes bundled with analysis software.

27. Applications
 For the evaluation of antiarrhythmics agents.
 In kidney cells.
 Used for isolated ventricular myocytes from Guinea pigs to study a
cardio selective inhibition of the ATP sensitive potassium channel.
 To identify multiple types of calcium channels.
 To measure the effect of potassium channel openers.
 Used in the molecular biology.

28.  Voltage clamp studies on sodium channels.
 Used to investigate a wide range of electrophysiological cell
properties.
 Measurement of cell membrane conductance.

29. THANK YOU