Biology Reference
In-Depth Information
purity. Freshly prepared dilutions of ATP and IP
3
(from frozen stocks) are added
to PS as required.
E
ering of the free [Ca
2
þ
], which might reasonably be varied between
nanomolar and several micromolar, requires bu
V
ective bu
V
V
Y
ers with appropriate a
nities for
Ca
2
þ
(
Patton
free [Ca
2
þ
]
1
m
M, BAPTA
et al., 2004
). For
less
than
(K
D
Ca
192 nM at pH 7.4) is preferable to EGTA because it has faster Ca
2
þ
-
binding kinetics and lesser pH-dependence. Where the free [Ca
2
þ
]ofPSis
1-100
m
M, we use 5,5
0
-dibromo BAPTA (K
D
Ca
1.83
m
M, Fluka), EGTA
(K
D
Ca
67 nM),
and/or
N-(2-hydroxyethyl)ethylenediamine-N,N
0
,N
0
-triacetic
acid (HEDTA, K
D
Ca
2.2
m
M, Sigma), alone or in appropriate combinations
(
Bers et al., 1994
). We initially estimate the amount of CaCl
2
required to achieve
-
mented with ATP, IP
3
, etc.) directly using either a fluorescent Ca
2
þ
indicator
(Fluo-3, K
D
Ca
325 nM, Invitrogen) or a Ca
2
þ
-sensitive electrode (Mettler
Toledo Ingold, Fisher Scientific) for higher free [Ca
2
þ
](
Dellis
¼
et al., 2006;
Rahman et al., 2009
).
The osmolarities of all solutions are adjusted to
290-310 mOsm kg
1
using
glucose and mannitol, and verified using a vapor pressure osmometer (Wescor,
Inc.). This is more important for recordings in the on-nucleus configuration than
for recordings from excised patches (
Fig. 2
C). PS is prepared to be slightly (
10%)
hypo-osmolar to BS to aid formation of giga-Ohm seals (
Hamill et al.,1981
). All
recording solutions are filtered using detergent-free 0.2-
m
m filters (Acrodisc
Ò
syringe filters, Pall Corporation) (
Ogden, 1994
). Fresh recording solutions (with-
out added IP
3
or ATP) are prepared monthly and stored at 4
C.
The presence within the nuclear envelope of other large-conductance cation and
Cl
channels (
Franco-Obregon et al., 2000; Marchenko et al., 2005; Mazzanti
et al., 2001; Tabares et al., 1991
) might potentially contaminate recordings of
nuclear IP
3
R. In practice, this appears not to be a significant problem. If such
problems should arise, they can be mitigated by replacing KCl in BS and PS with
cesium methanesulfonate (CsCH
3
SO
3
): Cs
þ
permeates IP
3
R but not K
þ
channels
(
Tovey et al., 2010
), while most anion channels are impermeable to CH
3
SO
3
.
D. Patch-Clamp Recording
The equipment required for nuclear patch-clamp recording is the same as that
used for conventional patch-clamp recording (
Fig. 1
A). The basic rig includes an
amplifier, headstage, electrode holder, micromanipulator, AgCl bath electrode,
data acquisition system (i.e., analog-to-digital converter, computer, and software),
inverted microscope, air table, and a Faraday cage. In addition, a pipette puller
and fire-polisher or microforge are required to fabricate electrodes. Optional
extras include systems for exchange of solutions, an oscilloscope, and a low-pass
8-pole Bessel filter; the latter extends the filtering range down to 0.1 Hz from the
1 to 100 kHz provided by the inbuilt filter. Comprehensive descriptions of the
