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The Whole-Cell Patch-Clamp Technique a powerful tool to approach CFTR function
Martin J. Hug:
1/9

12-01-2004

The whole cell patch-clamp technique
Provide through:

The European Working Group on CFTR Expression

The Whole-Cell Patch-Clamp Technique a powerful tool to
approach CFTR function

Author
: Martin J. Hug

University of Muenster, Institute of Physiology Nanolab,
Robert-Koch St. 55a, D-48149 Muenster, Germany

e-mail:
hugma@hugma.com


INTRODUCTION:

The patch-clamp technique provides a large array of different applications to assess
ion channel function. The activity and the gating behaviour of single channels are
best investigated using excised or cell-attached membrane patches. In many cases,
the likelihood of finding an active channel in the small area of membrane attached to
the pipette is unfortunately fairly low. A more integral approach is to study the
concerted action of many channels in the whole cell (1). Based on our knowledge
that CFTR is a protein that interacts with and is influenced by other ion channels, the
whole-cell (WC-) patch-clamp technique is ideally suited to improve our overall
understanding of CFTR function. Moreover, the preserved cellular compartment of
the whole cell provides all elements of the intracellular signaling cascade required for
the physiological regulation of the channel. It is also worthwhile to note that the WC-
mode was the first (and as yet only) variant of the patch-clamp technique to be used
in automated systems (2).

What is unique about the WC patch-clamp technique?

In principle the WC patch-clamp offers two different electrical parameters to be
measured: first, the membrane potential at a given current (V
m
) and second, the
current across the membrane at a given voltage (I). Sophisticated systems can
nowadays utilize these two parameters to derive additional electrical properties such
as membrane conductance and capacitance. Similar to the excised patch
configuration, the WC patch-clamp mode has the opportunity to control the
composition of the solutions on both sides of the cell membrane. The cytosolic
compartment is small compared to the volume of the patch pipette. Therefore, the
cellular interior becomes dialysed by the solution the patch pipette is filled with once
access to the cell is gained. It is reasonable to assume that within a few seconds the
concentrations of electrolytes within the cell equal those in the pipette. Addition of
modulators of cellular function to the pipette solutions permits the introduction of
these compounds into the cellular compartment. Normally the composition of the
pipette solution cannot be changed once intimate contact between the pipette and
the cell has been established. However, some investigators have developed means
to internally perfuse the patch pipette in order to change the pipette solution during
an experiment (3).

The bathing solution defines the extracellular compartment and thus allows for a
rapid change in the ionic composition outside the cell. To avoid mechanical
disturbances during the experiment utmost care needs to be taken to ensure that the
flow remains continuous once the perfusate is changed. Martin J. Hug:
2/9

12-01-2004

The whole cell patch-clamp technique
Provide through:

The European Working Group on CFTR Expression

Prerequisites for successful WC measurements are first, stable electrical access to
the cell interior and second, sufficient seal resistance to minimize leak currents. To
meet these requirements a tight seal needs to be established (see protocol D.1

Investigating the CFTR Cl
-
channel using the patch-clamp technique: An overview
;
(1)). The quality of the seal is subsequently estimated either by predefined
membrane test algorithms or by simply checking the signal to noise ratio. The next
and by far the most demanding step is to gain access to the cell:

There are three ways to yield sufficient electrical access to the cell:

1. Suction: The most frequently used approach is to rupture the membrane patch
underneath the patch pipette tip by brief suction using either a syringe or a
mouthpiece. Although this sounds a rather simple undertaking, it appears to be the
most difficult step in the entire experiment. Much care needs to be taken to ensure
that there is no movement of the pipette in the very moment suction is applied. To
prevent unwanted movement of the pipette, the pipette should be (i) exactly centered
and (ii) tightly mounted in the pipette holder. Even after successful rupture of the
membrane, a stable recording may not be achieved. Most often membrane debris
and cellular organelles creep into the mouth of the pipette and clog the small
aperture leading to loss of electrical access. Similarly, changes in cell volume during
the experiment can cause loss of electrical access bringing the recording to a
premature end.

2. Zapping: A feature offered by some patch-clamp amplifiers is the so-called zap
mode. The brief application of a strong voltage pulse (zap, ~ 1 V) across the small
membrane area underneath the pipette tip electroporates the patch thus removing
the electrical barrier. This approach largely depends on the cell type and does not
always yield sufficient access to the cell.

3. Permeabilisation: Addition of polyene antimycotics such as Nystatin or
Amphothericin B to the pipette solution introduces small, nonselective pores into the
membrane while the physical characteristics of the lipid bilayer remain intact (see
also protocol D.6
Measurement of apical CFTR Cl- currents in polarised epithelia
;
(4)). This approach is the least intrusive way to gain access to the cell when
compared to either suction or zapping. However, many cells possess a membrane
structure that prevents the use of this approach.

How can successful access to the cell be identified?

While all three approaches, suction, zapping and permeabilisation have their
caveats, they aim to achieve the same goal: electrical access to the cell. The
success of each approach can be identified by an increase in the current and the
appearance of small capacitative transients when voltage pulses are applied across
the membrane (see below). It is also possible to monitor a sudden change in the
apparent voltage caused by the membrane potential that can be measured as soon
as the inner surface of the membrane is electrically accessible.

Martin J. Hug:
3/9

12-01-2004

The whole cell patch-clamp technique
Provide through:

The European Working Group on CFTR Expression

G
a

C
p

C
m

G
m

Electrical equivalent of the WC configuration:

Once the WC mode is established, the patch pipette and cell form a complex circuitry
that we briefly need to examine. The schematic in figure 1 depicts the components
of this circuitry.









Patch
Pipette
Cell

Figure 1: equivalent circuit for the WC patch-clamp configuration.

All electrical parameters are measured through the resistive pathway of the access
conductance (G
a
= 1/R
s
). It is obvious that the larger G
a
or the smaller R
s
, the better
the signal to noise ratio. G
a
is shunted via the capacitative properties (C
p
) of the
patch pipette. C
p
depends on the size, shape and material of the patch pipette and
also on the height of the bath solution. It does influence the time-course and size of
the signal especially when small currents are measured. Most patch-clamp
amplifiers are therefore equipped with a circuit setting that permits the cancellation of
C
p
. The component that is most interesting to us is the membrane conductance,
G
m
(= 1 / R
m
). Similar to the access conductance, G
m
is also shunted by a capacitor,
the membrane capacitance C
m
. The latter is dependent on the membrane surface
area with a proportionality of 1 �F/cm
2
. While this feature can also be utilized to
study dynamic endo- and exocytotic processes in the plasma membrane it is mostly
used to standardize the measured whole cell current to a defined cell size (I/C
m
,
[pA/pF]).

It can easily be anticipated that G
a
and G
m
are two resistors in series that normally
cannot be measured independently. If a voltage U is applied across the pipette, the
cell and the bath, respectively, the resulting current I is defined by Ohms law:
I
=
U
R
s
+
R
m
(1)
If we use the terms G
a
and G
m
respectively, the equation reads:
I
=
U G
a G
m
G
a
+
G
m
(2)
We may now solve the equation for
G
m
=
I G
a
U G
a I
(3)
As in any circuitry the parameters depicted here influence the value of the voltage or
the current measured. Thus, proper analysis of the individual components of this Martin J. Hug:
4/9

12-01-2004

The whole cell patch-clamp technique
Provide through:
The European Working Group on CFTR Expression

network is necessary to interpret the data obtained with the WC patch-clamp
technique. It is therefore mandatory to repetitively measure G
a
during an experiment
in order to obtain correct values for G
m
.

Influence of capacitative components

Capacitative currents can be observed even before the WC configuration has been
established. When a voltage pulse is applied across a tight membrane seal, current
responses with a very short time course are noticed (Fig. 2A). The component
responsible for these tr