Biology Reference
In-Depth Information
Drifting baseline in current traces is first checked and corrected manually using
ClampFit. Current-amplitude histograms of recordings with no obvious sub-con-
ductance states are measured with a half-amplitude threshold-crossing criterion
using ClampFit (
Sachs et al., 1982
; Colquhoun, 1994). Only events lasting longer
than twice the filter rise time (t
r
¼
V
frequency of the
filter) can reach their full amplitude and so be reliably measured in a threshold-
crossing-based idealization procedure (Colquhoun, 1994) (
Fig. 4
A and B).
In recordings with 1-kHz filtering, the predicted filter rise time (assuming the filter
behaves as a Gaussian filter) is 332
m
s. Events lasting
0.3321/f
c
, where f
c
is the cuto
1 ms are, therefore, omitted
from the amplitude histograms. The peaks of the binned current amplitude histo-
grams are fitted in ClampFit by sums of the appropriate number of Gaussian
<
10 pA
A
C
100 ms
O
1000
C
500
O
B
0
C
0
5
10
Amplitude (pA)
D
0.06
t
c1
= 1.14 ms
(88%)
t
o
=10ms
0.06
0.04
0.03
t
c2
=92ms
(12%)
0.02
0
0
−
1
1
0
1
2
0
1
2
Log (duration, ms)
Log (duration, ms)
Fig. 4
Analysis of the behavior of single IP
3
R from nuclear patch-clamping recording. (A) Fragment
of a raw record from an excised lumen-out nuclear patch with a single functional IP
3
R3. The recording
conditions were identical to those shown in
Fig. 3
B. (B) The idealized record of the same trace produced
as described in the text (
Section IV.E
). This idealized record is used for all subsequent analyses. (C) All-
points current amplitude histogram, showing two peaks, one at 0 pA (the closed state) and a second at
10 pA (the single open state of one IP
3
R). (D) Distribution of the open (top) and closed (bottom) life-
times shown as Sigworth-Sine plots (
Sigworth and Sine, 1987
). The plots suggest a single open state with
t
o
of 10 ms, and two closed states with
t
c
of 1 and 92 ms.
