Biology Reference
In-Depth Information
equipment used for patch-clamp recording are presented in relevant chapters of
Sakmann and Neher (1995)
. Among the many steps taken to minimize electrical
noise, the following are particularly important: appropriate grounding of equip-
ment, use of thick-walled glass capillaries, filling pipettes with PS to the minimal
level required to contact the recording electrode, and minimizing immersion of the
pipette in BS (
Rae and Levis, 1992
).
Pipettes are pulled from filamented, thick-walled borosilicate glass capillaries
(GC150-10F, Clark Electromedical Instruments) using a Flaming/Brown P-87
horizontal micropipette puller (Sutter Instruments), and then fire-polished to a
tip diameter of
1
m
m using a microforge (MF-830, Narishige). It is advisable to
melt a small bead of glass onto the wire of the forge to prevent platinum vapor
from reaching the pipette tip. With monovalent cations as charge carriers, the
single-channel conductance (
g
)ofIP
3
R is large enough (
360 pS,
Section IV.E
)to
achieve good signal-to-noise ratios without hydrophobic coating of the patch-
pipette (
Penner, 1995
). But when
g
of IP
3
R is reduced, with pore mutants or with
Ca
2
þ
or Ba
2
þ
as charge carriers, for example, it may be necessary to coat pipette
tips with Sylgard
TM
(
Dellis et al., 2006, 2008
). When filled with PS, the pipette
resistance typically remains within the range of 15-20 M
O
. Pipettes are best
prepared a few hours before experiments. Unused pipettes can, however, be stored
in an air-tight container and used later, but it is advisable to repolish them lightly
before use to remove any impurities accumulated during storage.
Petri dishes are precoated with poly-
-lysine (0.01%, Sigma)
for 1-2 h, then rinsed twice with deionized water and air-dried. The nuclear
preparation (15
m
l) is added to a Petri dish containing BS (1.5 ml) and the cells/
nuclei are allowed to adhere. The dish is then mounted on the stage of an inverted
microscope (Zeiss Axiovert 100) coupled to an assembly of headstage (CV 203 BU,
Molecular Devices) and micromanipulator (PCS-1000, Burleigh Instruments).
Recordings are made at room temperature (
l
-ornithine or poly-
l
20
C) in the on-nucleus or excised
configuration (
Fig. 2
C). The latter is preferable because it allows control of the
medium on both sides of the membrane and e
V
ective control of the voltage across
the patch.
A nucleus largely free from debris is first identified (
Fig. 2
B) and the patch-
pipette is positioned, using the micromanipulator, with its tip just above the
nucleus. A slight positive pressure is applied to the inside of the patch-pipette
before dipping it into the BS to avoid dirt accumulating at the pipette tip and to
prevent backflow of BS into the PS (
Hamill et al., 1981
). After dipping the pipette
into BS, the pipette capacitance is compensated using the specific o
V
set on the
amplifier and the pipette resistance (typically
10-15 M
O
) is noted. As the pipette
tip approaches the nucleus, the positive pressure is relieved. Taking care not to
puncture the nuclear membrane, the pipette is lowered until it contacts the mem-
brane, which should increase the pipette resistance by at least 2 M
O
. A giga-Ohm
seal (
5G
O
) usually forms within a few seconds of applying slight negative
pressure, by suction, to the inside of the pipette; this is usually controlled by an
attached 50-ml syringe or by mouth. Seal formation can sometimes be facilitated
