Biology Reference
In-Depth Information
Analyses like these identify the numbers of stable open and closed states and
plausible relationships between them. They lead thereby to models of the steps
through which the IP
3
R passes between its inactive and open states. Such analyses
have so far been rather limited for IP
3
R, but they clearly suggest the existence of a
single open state and several closed states (
Ionescu et al.,2007;Rahmanet al.,2009
)
(
Fig. 4
D).
Extending the analysis to patches, in which we detected several IP
3
R, allowed us
to demonstrate that IP
3
causes IP
3
R to form small clusters of
4-5 channels
within which
t
o
is reduced from
5ms (
Rahman et al., 2009
). These
observations lead us to suggest that IP
3
contribute to the evolution of elementary
Ca
2
þ
signals by both regulating IP
3
R activity and by assembling IP
3
R into clusters,
within which regulation of IP
3
RbyCa
2
þ
and IP
3
is retuned (
Rahman and Taylor,
2009; Rahman et al., 2009; Taylor et al., 2009a
).
For most channels, including IP
3
R, single-channel open probability (P
o
) (rather
than
g
or the number of active channels) is the behavior that changes as the
stimulus intensity varies. Increasing IP
3
or Ca
2
þ
increases P
o
of IP
3
R because
both ligands shorten the duration of the closed times, without a
10 to
ecting
t
o
; hence,
the probability of finding the channel open (P
o
) is increased (
Foskett et al., 2007;
Rahman et al.,2009
). P
o
is calculated from the fitted amplitude histograms of the
current traces (typically lasting
V
1 min for IP
3
R) (
Ding and Sachs, 1999
):
A
o
A
o
þ A
c
P
o
¼
ð5Þ
where A
o
and A
c
are the areas under the curves corresponding to the open and
closed states in the current amplitude histogram, respectively.
When IP
3
R activity is low, it becomes very di
cult to know how many channels
are contributing because it is unlikely that all will open simultaneously. Under
these conditions, the overall activity is better expressed as NP
o
which is defined as
(
Ching et al., 1999; Rahman et al., 2009
):
Y
N
1
ntðÞ
T
n¼
NP
¼
ð6Þ
o
where t
n
is the total time for which n IP
3
R are simultaneously open and T is the
duration of the recording.
V. Concluding Remarks
Patch-clamp recording of IP
3
R expressed within the nuclear envelope allows
single-channel analyses of these otherwise inaccessible intracellular Ca
2
þ
channels
(
Figs. 1 and 2
). DT40-KO cells provide a null background (
Fig. 3
) for expression of
recombinant and mutant IP
3
R allowing functional analysis of IP
3
R with defined
