Analytical Methods in Biomedicine---Master Program Hochschule Reutlingen (Reutlingen University)

1. Vorname Nachname, Funktion, Bereich, Hochschule Reutlingen, Alteburgstraße 150, 72762 Reutlingen, www.reutlingen-university.de,
T. +49 (0)7121 271-Durchwahl, F. +49 (0)7121 271-Durchwahl, vorname.nachname@reutlingen-university.de
PATCH CLAMP TECHNIQUE
Aqif Siddique
26-11-2015
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2. Table of Contents
Introduction
History
Principle
Configuration & types
Application
Limitation
Recent research
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3. Introduction
Patch clamp is a laboratory technique in electrophysiology,
that allows the study of single or multiple ion channels in
cells.
Sakmann and Neher develop this technique in 1970s and early
1980s.
Received Nobel prize for this high scientific work in 1991.
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4. Historical Development
Jan
Swammerdam
• Earliest
experiments
in electro-
physiology
Luigi Galvani
• First experimental
evidence of
electrical activity
in animals by
using metal wires
in frog muscle
Hodgkin &
Huxley
• First
intracellular
measurement
of action
potential in the
giant squid
axon
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5. Graham
• Impaling
micropipettes
developed by
skeletal
muscle fibers
Cole &
Marmont
• Voltage clamp
technique
combined with
micropipettes
Sakmann &
Neher
• patch clamp
technique
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6. The patch-clamp technique
Erwin Neher
Bert Sakmann
Germany
(1991 Nobel Laureates)
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7. Need for patch clamp
Patch clamp is refinement of voltage clamp technique.
Provides low-noise recordings of current.
Provides access to the inside of the cell.
Can insert an electrode into the cell
Can change the intracellular fluid
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8. Creates a seal impermeable to ion flow!!!
High electrical resistance
Allows one to measure current through ion channels vs.
voltage, time, temperature
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9. Basic Principle
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
Fire -polished glass pipette
Electrolyte solution
Electrode (10-25 µm)
<10nm
10 GΩ resistor at 20°C, the standard
deviation of the current noise at 1
kHz will be 0.04 p-A,
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10. 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.
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11. Configuration of patch clamp
 On cell
 Inside Out
 Whole Cell
 Outside Out
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12. Variations
Cell-attached or on-cell patch
Inside-out patch
Whole-cell recording or whole-cell patch
Outside-out patch
Perforated patch
Loose patch
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13. Cell-attached or On-cell Patch
The electrode is sealed to the patch of membrane and cell
remains intact.
This allows 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, 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.
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14. Inside-out Patch
After the gigaseal is formed, the micropipette is quickly
withdrawn from the cell.
Thus ripping the patch of membrane off the cell, leaving the
patch of membrane attached to the micropipette, and exposing
the intracellular surface of the membrane to the external
media.
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15. This is useful when an experimenter wishes to manipulate the
environment at the intracellular surface of ion channels.
For example, channels that are activated by intracellular
ligands can then be studied through a range of ligand
concentrations.
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16. 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.
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17. Whole-cell Patch
Disadvantage
Volume of electrode is larger
than cell, so the soluble
contents of the cell's interior
are slowly replaced by
contents of electrode. This is
referred to as electrode
"dialyzing” the cell's contents.
Advantage
Large opening at the tip of
the patch clamp electrode
provides lower resistance
and thus better electrical
access to the inside of the
cell.
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20. Patch clamp technique in isolated
cardiac myocytes
Perfusion of a section of intact
canine left ventricular myocardium.
A cannula has been placed into the
left anterior descending coronary
artery and clamps have been placed
to occlude major coronary artery
branches that have been transected
during sectioning.
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21. Limitations
oImparting skillful training performance and recording In
during single channel recordings
oCost of process is expensive
oTime consuming
oNumber of samples required is more at times
oChance of membrane distortion
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22. Applications
 For the evaluation of anti arrhythmic 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.
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23. To measure the effect of potassium channel openers.
Used in the molecular biology.
Voltage clamp studies on sodium channels.
Used to investigate a wide range of electrophysiological
cell properties.
Measurement of cell membrane conductance.
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24. Recent research
Measurements are conducted in a multi-parametric manner in
an integrated and automated microfluidic chip.
Micropipettes in traditional patch clamp technique are replaced
by nano machine patch clamp system with integrated micro
fluidics which aids:
o Rapid Intra cellular perfusion
o Improved optical measurements
o Rapid measurement of single cell dose response curves
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25. Conclusion
It is highly modified and successful technique
Development of this technique is being done for newer
approaches to yield better accurate and efficient information
which aids drug discovery process.
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26. References
• Wyllie DJA (2007) Single-channel recording. In Patch-
Clamp Analysis – Advanced Techniques 2nd Edition. pp 69-
129. Ed. Walz W. Humana Press Totowa, New Jersey USA
• Sakmann B (1992) Elementary steps in synaptic
transmission revealed by currents through single ion
channels. Neuron 8, 613-629.
• Aidley DJ & Stanfield PR (1996) Ion Channels – Molecules
in Action Cambridge University Press.
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