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probability density functions (pdfs) (
Fig. 4
C). Mean current amplitudes are plotted
against the corresponding applied potentials to create I-V curves (see
Fig. 5
).
The unitary conductance (
g
) and reversal potentials (E
rev
) are derived by
linear least-square regression analysis using statistical package such as Prism 5
(GraphPad Software, Inc.) or Origin Pro 7.5 (OriginLab Corporation).
Because
g
is a fundamental property of any ion channel, reflecting interactions
between permeating ions and the residues that form the pore and lead to it, I-V
relationships are often used as ''fingerprints'' to help identify channels. With K
þ
as
charge carrier, the I-V relationships of mammalian IP
3
R in excised nuclear patches
from DT40-KO cells are linear across a range of applied potentials (
60 to
þ
60 mV) (
Fig. 5
). From the slopes of these I-V plots, we have consistently
observed two populations of IP
3
R with unitary K
þ
conductances (
g
K
) of either
200 pS (
Dellis et al., 2006; Rahman and Taylor, 2009; Rahman et al.,
2009
)(
Fig. 5
B). Neither current was detected in the nuclear envelope of DT40-KO
cells or from DT40-KO cells stably expressing IP
3
R in the absence of IP
3
,orwith
IP
3
in the presence of a competitive antagonist. These values of
g
K
are lower than
reported (
120 or
320-360 pS) for IP
3
R in the nuclear envelope of mammalian cells
(
Foskett et al., 2007
), but there is wide variation in published values (from
9to
480 pS) (
Cheung et al., 2010; Rahman and Taylor, 2009
). The reason for these
disparities is unresolved, but it may reflect variable amounts of free Mg
2
þ
in PS
causing a reduction in
g
K
(
Mak and Foskett, 1998; Rahman and Taylor, 2009
).
It is, however, clear from analyses of I-V relationships that all IP
3
R have a large
g
for monovalent cations and lesser
g
for bivalent cations (
Dellis et al., 2006; Foskett
et al., 2007
)(
Fig. 5
B).
A
B
20 pA
15
I
(pA)
500 ms
10
+60 mV
C
5
C
+40 mV
−
80
−
40
40
80
5
−
1
−
V (mV)
0mV
C
−
40 mV
C
−
15
−
60 mV
C
Fig. 5
Current-voltage relationship for nuclear IP
3
R. (A) Currents were recorded from lumen-out
patches excised from the nucleus of DT40-KO cells stably expressing IP
3
R3. PS included IP
3
(10
m
M),
ATP (5 mM), and a free [Ca
2
þ
]of
200 nM. K
þ
was the charge-carrier and the holding potential was
varied between
þ
60 and
60 mV as shown. C denotes the closed state. (B) From the slope of the
current-voltage (I-V) relationship,
g
was 208 pS. Results are means
SEM, n
¼
4.
