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containing some isolated nuclei is stored in NIM on ice and used within 1 h for
patch-clamp experiments. Because the activity of nuclear IP 3 R has been reported
to decrease after
40 min at 20 C( Boehning et al., 2001a ), we routinely prepare
fresh nuclei at hourly intervals. DT40 cells are not much larger than their nuclei
( Fig. 2 B). The inexperienced eye may therefore find it di
Y
cult to distinguish nuclei
from cells. But nuclei rarely have the smooth surface of intact cells and they often
only partially protrude from broken cells, where the relatively clean exposed
surface allows formation of a giga-Ohm seal. The yield of nuclei can be substan-
tially increased to 50-60% using methods that require incubation in hypo-osmolar
media ( Franco-Obregon et al., 2000 ), but we rarely detect active IP 3 R after such
isolation procedures. A nuclear isolation kit (Sigma Nuclei EZ Prep) also provides
nuclei in high yield (
85%), but we rarely succeed in forming giga-Ohm seals with
these nuclei. In practice, the low yield of nuclei with our protocol is not a limitation
for nuclear patch-clamp recording.
IP 3 R have also been reported to be expressed within the inner nuclear membrane
( Humbert et al., 1996; Marchenko et al., 2005 ). The citrate treatment used to
remove the outer nuclear membrane and so allow patch-clamp recording of
IP 3 R within the inner membrane ( Marchenko et al., 2005 ) appears not, at least
in our experience, to be readily applicable to DT40 cell nuclei.
C. Solutions for Patch-Clamp Recording
For most recordings, we use K þ as the charge-carrier. This eliminates the
complexity of having Ca 2 þ passing through the IP 3 R regulate its activity, and it
provides larger single-channel currents than with bivalent cations ( Rahman et al.,
2009 ). The bath solution (BS), which bathes the luminal surface of the nuclear
envelope, typically contains 140 mM KCl, 10 mM HEPES, 100 m M 1,2-bis(2-
aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid (BAPTA, tetra potassium salt,
Calbiochem), and a free [Ca 2 þ ]of
200 nM (total CaCl 2 ,51 m M) adjusted to pH
7.1 with KOH. The usual pipette solution (PS), which bathes the cytosolic surface
of the membrane, contains 140 mM KCl, 10 mM HEPES, 500 m M BAPTA,
Na 2 ATP (0.5 mM), IP 3 (American Radiolabeled Chemicals, Inc.), and a free
[Ca 2 þ ]of
254 m M) adjusted to pH 7.1 with KOH.
IP 3 ,Ca 2 þ , and ATP are the three ligands of IP 3 R whose concentrations must be
adjusted to obtain optimal IP 3 R activity in patch-clamp recording ( Foskett et al.,
2007 ). The concentration of IP 3 in PS can be varied between experiments, depend-
ing on the aim of the analysis; 10 m Mwill usually be su
200 nM (total CaCl 2 ,
cient to saturate responses
to IP 3 ( Foskett et al., 2007; Rahman et al., 2009 ). The potentiating e
Y
V
ects of ATP
di
er between IP 3 R subtypes with higher concentrations required optimally to
activate IP 3 R3 ( Betzenhauser et al., 2008a; Miyakawa et al., 1999 ). The pH of PS
must be readjusted after addition of ATP, and its e
V
ects on free [Ca 2 þ ] also need to
V
be considered. Finally, because Mg 2 þ a
ects the conductance of IP 3 R( Mak and
Foskett, 1998; Rahman and Taylor, 2009 ), it is advisable to use ATP of the highest
V
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