Biology Reference
In-Depth Information
by applying a holding potential of about
40 mV once a high-resistance contact is
established (
Ogden, 1994
), and occasionally giga-Ohm seals form spontaneously.
Seals
5G
O
can be routinely obtained with mild suction providing the nucleus is
immobile and free of debris.
To forman excised patch, our preferred recording configuration, the patch is pulled
from the nucleus after forming the giga-Ohm seal (
Fig. 2
C). To prevent formation of
closed vesicles at the tip of the patch-pipette, excisedpatches are briefly (1-2 s) exposed
to air and then reimmersed in BS (
Hamill et al.,1981
). Prewritten protocols are then
used to record currents through the excised patch at di
>
erent holding potentials. The
bath electrode is grounded (i.e., 0 mV) and for convenience, the potential across a
nuclear patch (whether attached or excised) is defined as the pipette potential minus
the bath potential. That is, with symmetrical media, a positive holding potential would
favor movement of cations from PS (the cytosolic surface) into BS (the luminal
surface) producing an outward current and an upward deflection on the channel
record (
Fig. 2
DandE)(
Franco-Obregon et al., 2000; Mak and Foskett, 1994;
Rahman et al.,2009
). For determination of current-voltage (I-V) relationships, and
thereby the single-channel conductance (
g
) of the channel (
Section IV.E
), the voltage
across the excised patch can be stepped from
V
60 mV in increments of 20 mV
froma holding potential of 0 mV. Applyingmore extreme voltages a
60 to
þ
ects the stability
of the nuclear patch. For all other experiments, including kinetic analyses, currents are
typically recorded at
V
40 mV for between 1 and 10 min.
Currents are amplified with an Axopatch 200B amplifier in its voltage-clamp
mode, filtered at 1 kHz with a low-pass 4-pole Bessel filter (built into the amplifier),
and digitized at 10 kHz with a Digidata 1322A interface using the PC-based
acquisition software package pClamp 9.2 (Molecular Devices) (
Colquhoun,
1994
). This filtering, while it inevitably causes some loss of information, has the
e
þ
ect of rejecting signals (background noise) that are too brief to reflect the gating
of IP
3
R. If the filtering frequency is set too low, it will reject events that do reflect
gating of channels, and if set too high, background noise will obscure the openings.
The optimal filtering frequency is, therefore, a compromise that depends upon the
noise and time-course of the channel events; it needs to be optimized empirically.
The sampling rate must, of course, exceed the filter frequency if further valuable
information is not to be lost as the signals are digitized. In practice, digitization
should be 10-20 times faster than the cuto
V
or ''corner'' frequency of the filter
(Colquhoun, 1994). Most nuclear patch-clamp studies of IP
3
R have used 1-kHz
filtering (
Dellis et al., 2006; Ionescu et al., 2006; Mak and Foskett, 1997; Rahman
et al., 2009
). For presentation, traces can be further filtered o
V
Z
ine using a Gaussian
filter (built within ClampFit).
For the determination of relative permeabilities to cations, asymmetric record-
ing solutions are used. For example, the normal BS can be replaced by a Ba
2
þ
-rich
BS (50 mM BaCl
2
, 30 mM KCl, 10 mM HEPES, adjusted to pH 7.1 with KOH),
while the PS remains unchanged (
Boehning et al., 2001a; Dellis et al., 2006
). The
liquid junction potential (LJP) under this asymmetric condition can be predicted
(5.2 mV at 20
C), using the ''junction potential calculator'' (JPCalc, within
