The document discusses the patch clamp technique, which allows the study of single or multiple ion channels in cells. It was developed in the late 1970s/early 1980s by Erwin Neher and Bert Sakmann, who received the Nobel Prize for this work. There are different configurations of patch clamp including cell-attached, whole-cell, outside-out, and inside-out. The technique involves pressing a glass pipette against a cell to form an electrical seal and record currents. It has applications in studying ion channels in excitable cells and the effects of drugs.

1. KASHMEERA N.A.
ROLL NO: 37
CHRIST COLLEGE, IJK

3. PATCH CLAMP RECORDINGPATCH CLAMP RECORDING
 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

4. •Erwin Neher and Bert Sakmann developed the patch clamp in the
late 1970s and early 1980s. They received the Nobel Prize in
Physiology or Medicine in 1991 for this work.

5. THE PATCH-CLAMP TECHNIQUE
Erwin Neher
Bert Sakmann
Germany
(1991 Nobel Laureates)
5

7. BASIC 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

8. TYPES OF PATCH CLAMP:
 Cell attached
 Inside Out
 Whole Cell
 OutsideOut

9. The patch-clamp technique can be applied
in several configurations
‘CELL-ATTACHED’

10. The cell-attached recording mode is the first step necessary for
establishing any other patch-clamp configuration.
In order to form the cell-attached mode, a pipette tip is placed on the
surface of the cell, forming a low resistance contact (seal) with its
membrane.
Slight suction applied to the upper end of the pipette results in
formation of a tight seal with a resistance of 1 to 100 Gigaohm. Such
a seal with a resistance in the range of gigaohms is called “giga-seal”.
Formation of a giga-seal is extremely important for reduction of noise
during single-channel recordings.
CELL-ATTACHED

11. ‘WHOLE-CELL’

12. Whole-cell recordings 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.
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 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. Thus, any properties of the cell that depend on
soluble intracellular contents will be altered. Generally speaking, there is a
period at the beginning of a whole-cell recording, lasting approximately 10
minutes, when one can take measurements before the cell has been dialyzed.

13. ‘OUTSIDE-OUT’

14. 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.
. 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

15. ‘INSIDE-OUT’

16. By quickly withdrawing the electrode from the cell after obtaining a
cell attached configuration, the patch of membrane within the tip
of the electrode can be torn from the cell while maintaining a
gigaohm seal with the electrode. This configuration is referred to
as an inside-out patch, in which the interior aspect of the cell
membrane is exposed to the bath solution and the exterior of the
membrane is exposed to the internal pipette solution. The inside
-out patch configuration is useful for studying the effects of
manipulating the internal environment on single ion channel
function.

17. The patch pipette with internal recording electrode (A) and reference electrode
(B) are connected to the headstage(C), which is mounted on
a micromanipulator (D). Isolated cells are visualized with an inverted light
microscope (E). The microscope, micromanipulator,
and headstage are placed on a air table (F) to isolate these components from
vibrations that may interfere
with gigaseal formation, and placed within a Faraday cage (G) to shield the
setup from ambient electrical noise. The
acquired analog signal from the headstage is passed to an analog to digital
converter (H), where the signal is digitalized
and sent to a computer (I) for data analysis. An oscilloscope (J) is used for
monitoring experiments and for data display.
Experimental
set up

20. Air table: Uses pressurized cylinders to ‘‘float’’ the table and
isolate the preparation being studied from vibrations that
can interfere with the ability to achieve high resistance pipetteemembrane
seals.
Amplifier: Amplifies current or voltage being measured and interfaces
with computer for data acquisition. Also controls
membrane voltage (voltage clamp) or current (current
clamp) depending upon experimental protocol.

21. Analog to digital converter: Converts the analog signal recorded
by the microelectrode to digital data that is acquired by
computer during experiments.
Computer: Uses any of a number of proprietary software packages
for data acquisition and analysis. These suites allow the
experimenter to control membrane voltage/current, to process the input from
the amplifier (filtering, capacitance
compensation, etc.), to apply stimulus protocols, and to analyze
acquired data.

22. Faraday cage: Shields the headstage from stray electromagnetic
fields that may introduce noise into electrophysiologic
recordings. Ideally composed of a copper mesh, the Faraday
cage is most effective when connected to a ground source.
The microscope and headstage are most commonly placed
inside the Faraday cage.
Headstage: The physical connection between the preparation
being studied and the amplifier, the headstage has inputs
for the recording microelectrode (which comes into physical
contact with the preparation) and ground wire (usually in the
external bath) and interfaces with the amplifier for data acquisition,
control of membrane voltage/current, and stimulation
of preparation when necessary. The headstage is mounted on
a micromanipulator.

23. Inverted microscope: Used to visualize cells/preparation being
studied. This is important in order to identify viable cells
and to assist with appropriate orientation of microelectrode
and cell to optimize high resistance seal formation.
Micromanipulator: Used to manipulate the microelectrode
(mounted on the headstage) to contact the cell/preparation
being studied. Micromanipulators may be mechanical, electrical
or hydraulic.

24. Figure 1. Registration of the flow of current through single ion
channels using the recording technique of Neher and Sakmann. A
schematically shows how a glass micropipette is brought in contact
with the cell, and B, using a higher magnification, a part of the cell
membrane, with ion channels, in close contact with the tip of the
pipette. The interior of the pipette is connected to an electronic
amplifier. C shows a channel in greater magnification with its
receptor facing the exterior of the cell and its ion filter. D shows the
current passing through the ion channel as it opens.

26. PATCH CLAMP TECHNIQUE IN ISOLATED CARDIAC
MYOCYTES
APPLICATIONSAPPLICATIONS
The generation of an action potential in heart muscle
cells depends on the opening and closing of ion-selective channels in
the plasma membrane.
 The patch-clamp technique enables the investigation of drug
interactions with ion-channel .
 The Isolated cells are ready for experiment.
 Glass micro-pipette - a tip opening of about 1 μm, is placed onto
the cell.

27. The patch-pipette is filled with either high NaCl or KCl
solution and is mounted on a micro manipulator.
A chlorided silver wire connects the pipette
solution to the head stage of an electronical amplifier.
A second chlorided silver wire is inserted into the bath
and serves a ground electrode.
Whole cell patch clamping is done.
 This high input resistance enables the recording of
small electrical currents in the range of Picosiemens
(10–12 S), which are flowing through channel-forming
proteins situated in the membrane patch.
The electrical current is driven by applying an electrical
potential across the membrane patch, and/or by
establishing an appropriated chemical gradient for the
respective ion species.

28. To investigate the interaction of drugs with all ion
channels involved in the functioning of the heart muscle
cell (K+, Na+, Ca2+ and eventually Cl– channels).
Concentration-response curves of drugs which either
inhibit or activate ion channels can be recorded either
on the single channel level or by measuring the wholecell
current.

30. Novel Ion Channels Can Be Characterized
by a Combination of Oocyte Expression
and Patch Clamping
Cloning of human disease-causing genes and sequencing of
the human genome have identified many genes encoding channel proteins,
including 67 K channel proteins.
One way of characterizing the function of these proteins is to transcribe a
cloned cDNA in a cell-free system to produce the corresponding mRNA.
Injection of this mRNA into frog oocytes and patch-clamp measurements on
the newly synthesized channel protein can often reveal its function .
This experimental approach is especially useful because frog oocytes
normally do not express any channel proteins, so only the channel under
study is present in the membrane. In addition, because of the large
size of frog oocytes, patch-clamping studies are technically
easier to perform on them than on smaller cells.

31. Here's what the results look like.
Time is recorded along the horizontal axis, and current along the vertical.
When the channel is closed, the current is 0; when it is open, it jumps up to a
tiny, fixed value. There is a quantal nature to the current flow at this level, as a
single channel allows a fixed number of ions to move through it per unit time,
just as opening a faucet tap allows only a certain volume of water to flow. We
can see that the transition state for an ion channel between open and closed
is very brief; it flicks open and closed quickly