Introduction to Patch
Clamp Technique
Robert H. Chow
Physiology & Biophysics
ZNI 325
rchow@usc.edu
Transport Across Cell Membranes
Cell Membrane

Delimits cell boundary -> defines cell
~ 4-5 nm
http://upload.wikimedia.org/wikipedia/commons/f/f0/Lipid_bilayer_section.gif
Transport Across Cell Membranes
Cell Membrane

Membrane is relatively impermeable to charged
molecules and ions

Introduces challenge of how to bring things into
and out of cell

Transmembrane transporters




Channels: ions, water, other
Transporters (passive and active): glucose, amino acids, other
Exocytosis/endocytosis
Invention of Patch Clamp Technique

Enabled measurement of ion currents through single
channels
Ion Channels



Transmembrane proteins
Gated pathway for ions to enter/exit cell
History of channelology
Concept: Hodgkin/Huxley (Nobel ‘70)
 Noise recordings, Katz (Nobel ‘72)
 Pore block/gating currents, Armstrong (Lasker ‘99)
 Size exclusion, Hille (Lasker ‘99)
 Single-channels, Neher & Sakmann (Nobel ‘91)
 Cloning, Numa
 Crystallization, Mackinnon (Lasker ‘99, Nobel ‘03)

What is "patch clamp"?




One of most powerful biophysical methods
today
Allows real-time monitoring of single protein
behavior in cell membranes -- ionic channels
Many other capabilities, too (see below)
Nobel Prize in Physiology & Medicine to
Neher & Sakmann, 1991
Patch Clamp Technique



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
Hollow glass pipette
Melted & pulled to produce tapering tip of
diameter 1-3 µm
Filled with electrolyte solution
Mounted in special pipette holder on
manipulator
Pipette pressed gently against cell membrane
Gentle suction applied to form “gigaseal”
http://www.science-display.com/new/datenbank/detail.php?action=edit&id=23
Glass-membrane seal
Note:
shape
Show pipette
holder
Modified from Sakmann and Neher, Sci. Am. 1991
Early Single-Channel
Recording
AChR in muscle
Neher, Nobel Lecture
cell-attached
inside-out
patch
whole-cell
outside-out
patch
Neher, Nobel Lecture
Ion Channels Are Gated Pores That Are
Selective For Transported Ions
Ligand-gated ion channel
Voltage-gated ion channel
Note: All-or-none openings, stochastic
From Ion Channels and Disease, Frances M. Ashcroft, Academic Press, 2000
Electrophysiology Conventions

Current is plotted as positive (upward) for
outward positive charge movement (or
inward negative charge movement).

Current is plotted as negative (downward)
for inward positive charge movement (or
outward negative charge movement).
Channel Names
Ligand-gated channels
•
Historically named after ligand
e.g. Acetylcholine receptor/channels,
glutamate receptor/channels
Voltage-gated channels
•
Historically named after permeating ion
e.g. Sodium channels, potassium
channels, calcium channels
Consensus names now based on DNA
sequence
Ion Channel Recordings

Microscopic
Ion currents through single channel or few
channels
 Typically recorded using patch clamp with
excised or isolated patch technique


Macroscopic
Ion currents through many channels
 typically recorded from entire cell or large
membrane patch with microelectrode or patch
clamp whole-cell technique

Macroscopic
Whole-cell
Microscopic
Single-channel
Sakmann, Nobel Lecture
Opening of voltage-gated ion channels is a stochastic process
Hille, Ionic Channels of Excitable
Membranes, 2nd ed., Sinauer
Associates, 1992
Ion Channel Analysis
Definitions
Recall:
Membrane potential
Vm = yi-yo
Vm = yi when yo = 0
Nernst Potential
membrane potential at which
membrane potential gradient = concentration gradient
So, net current is zero for a given ion type at ~Nernst potential
Ex=RT/ZF ln[x]o/[x]ί = 60 log10 [x]o/[x]ί
How to study a specific ion
channel type
1.
2.
3.
4.
remove ions of other types
increase concentration gradient of ion
of interest
toxins/drug blocker of other channels
current enhancer of channel of interest
Single-Channel Conductance
AChR channel
Step size (pA)
Plot of current size
as function of voltage
Slope = I/V = conductance
Sakmann, Nobel lecture
i=γ(Vm-Ex)
Ohms law for a single ion channel
γ is single channel conductance
and
Vm-Ex = “Force” that drives
ion movement
micro
i=γ(Vm-Ex)
macro
Im= npi
Ion current through membrane
Im , the whole cell current
i , current through a single ion channel
n , number of ion channels in membrane
p , probability of ion channel being open
γ , single channel conductance
Ex , Nernst Potential
Comparing Macroscopic and
Single-Channel Recording
Macroscopic
Whole-cell
Im= npi
Microscopic
Single-channel
i=γ(Vm-Ex)
Properties of Ion Channels 1

Gating and Selectivity

Gating
 Unlike
resistors, channels are open in an
all-or-none fashion
 Gating triggered by one of the following
 Voltage
change
 Chemical binding (e.g. transmitters)
 Mechanical force (e.g. stretch)
 Light
Gating is stochastic
Sakmann Nobel Lecture
Voltage-dependent gating is based on
movement of charges in the channel protein
Bezanilla, 2000. Physiol. Rev. 80(2):556
http://www.anes.ucla.edu/%7Epancho/MODEL/SHKMODEL.HTML
Bezanilla Model
S4 rotates, moving
+ charges from crevice
facing inwards to
another crevice
facing outwards
12 electron charges
move in the process
Movement of channel gates is seen
as“gating current”
Bezanilla, 2000. Physiol. Rev. 80(2):556
Ball-and-Chain Model of Inactivation
Hille, Ionic Channels of Excitable
Membranes, 2nd Ed., p. 356 & p. 357
Ashcroft, Ion Channels and
Disease, p. 107
Ball-and-Chain Model of Inactivation
Properties of Ion Channels 2

Selectivity
Channels not passive conduits
 Interact specifically with permeating ions
 “Select” ions that can go through
 Yet, allow high throughput (~1e6 ions/s)
 Channels named based on ion selectivity


e.g. Sodium channels, potassium channels, calcium
channels, etc.
Ion Channels as Molecular
Sieves
Li+
Na+
K+
Tl+
From Hille, Ionic Channels of Excitable Membranes, 2nd Ed., p. 356 & p. 357
Armstrong and Mackinnon on potassium
channel ion selectivity mechanism
ions
K
Na
Optimal size
Nonoptimal
water
“selectivity filter”
Ion fully dehydrated
Ion not fully dehydrated
Net energy change = dehydration + coordination
Xstall Structure 1
http://www.nature.com/nature/focus/ionchannel/
Xstall Structure 2
Fab fragments
Red = Selectivity filter
Mackinnon, Nature 2001
Green = K+
Red = water
OTHER APPLICATIONS OF PATCH CLAMP
1. “sniffer patch” for monitoring transmitter release
2. “single-cell biochemistry”: control of intracellular composition
introduction of chemicals/biological agents, etc.
e.g.
antibodies,
metabolic poisons,
protein fragments
diffusional removal of intracellular contents
e.g.
to evaluate role of 2nd mess.,
specific proteins
3. single-cell molecular biology
mRNA amplification & PCR
4. single-cell ionic/metabolic probes
fluorescent dyes
e.g. ion-sensitive dyes (FURA2, INDO, etc.)
5. Single-cell membrane capacitance measurements for exocytosis and
endocytosis
FURA-2: Example of calcium
ion reporter
Ratiometric dye, pathlength independent
(Roger Tsien)
Combined current and calcium
measurement
Macroscopic
Whole-cell
F390
F340
[Ca]
Alternately excite at 390 and 340 nm
While measuring fluorescence at ~510 nm
Calculate free [Ca], based on ratio of
F340/F390 (or other suitable wavelengths)
Single Cell Biochemistry /
Molecular Biology
1. Record/Image
2. RNA Processing
3. miRNA and
cDNA profiling
Cell
Patch clamp
e.g. membrane
current
or calcium
measurement
Total RNA
extraction &
amplification
miRNA &
cDNA
microarrays
Cell membrane is a capacitor

()
ee0
Cm =
A
d
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e = dielectric constant
e0 = polarizabilty of free space
d = membrane thickness
Cells have lipid plasma
membranes
Lipids are insulators
Cytoplasm and extracell
fluid are electrolytes
capacitance is proportional
to the surface area of the
insulator
By measuring the
capacitance we can
measure the surface area of
the cell.
Specific membrane capacitance

The proportionality factor is also called:
specific membrane capacitance:
0.7-1µF/cm2 or 7 - 10 fF/µm2


cell (12µm diameter) = 3.6 pF (10-12 F)

secretory granule (200 nm diameter) = 1 fF (10-15 F)

synaptic vesicle (50 nm diameter) = 60 aF (10-18 F)
Specific membrane capacitance is remarkably
constant among different cell types and even
different species.
Why measure capacitance?

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Surface area increases upon exocytosis,
due to addition of vesicle membrane to
plasma membrane
Monitoring surface area offers real-time
assay of exocytosis
Secretory cell ultrastructure:
Secretory machine
• Large Dense-Core Granules
about 10 - 30,000
diameter ~350 nm
Exocytosis: the Movie
Science Magazine Cover
1993, vol. 258
From
Molecular Cell Biology, 4th
Ed., edited by Lodish et al.
Whole-Cell Patch Clamp:
Three-component model
Frequency domain
Sine wave stimulus
Phase-sensitive Detector
Y( t)= ReY( t)+ ImY( t)
Ra
Cm
Rm
Time domain
Step function
C=Q/V,
Where Q= ∫ I dt
Calculating Cm in Time Domain
Vout
If Rm >> Ra
Ra
Vcom
Cm
Rm
Vcom
Vm
Im
Vm
Vcom
Vm
Recall Q=CV
C = Q/V, where
Q=
∫
I dt
The Cm Response to 6 Depolarizations
Black = control
Red = complexin KO
* p<0.05; ***p<0.00001
2 animals, 20 traces from 9 control cells
2 animals, 22 traces from 11 KO cells
END