Biology Reference
In-Depth Information
A
B
5pA
+40 mV
500 ms
5
I
(pA)
C
+60 mV
−
80
−
40
40
80
C
V (mV)
−
60 mV
C
−
5
Fig. 6
Determining the cation-selectivity of IP
3
R fromnuclear patch-clamp recording. (A) Currents were
recorded in the same way as described in
Fig. 5
A, but with the usual PS changed to include Ba
2
þ
(50 mM)
rather thanK
þ
. The currents recorded at di
V
erent holding potentials are shown. C denotes the closed state.
(B) I-V relationship showing a reversal potential (E
rev
)of
1.4 mV after correction for the liquid
junction potential (
Section IV.D
). From the modifiedGHK equation, this suggests that the permeability to
Ba
2
þ
relative to K
þ
(P
Ba
/P
K
) is 4.7. The unitary conductance (
g
) from the slope of the plot is 45
23.8
4pS.
absolute temperature (K). These analyses have established that IP
3
R(
Dellis et al.,
2006; Foskett et al., 2007
), like ryanodine receptors (
Williams, 2002
), are far less
selective (P
Ba
/P
K
7) than Ca
2
þ
channels in the plasma membrane (
Fig. 6
). The
distinction is important because channels within the plasma membrane must be
able to discriminate between the many ions with an electrochemical gradient across
the membrane, whereas Ca
2
þ
is probably the only cation with an appreciable
gradient across the ER membrane (
Somlyo et al., 1977
).
In addition to revealing the properties of the open pore, single-channel analyses
can also shed light on the steps that lead to its opening. The kinetic analyses of single-
channel records described here require that channel behavior has attained a steady-
state. This is most easily assessed from a stability plot of single-channel open
probability (P
o
) versus time (Colquhoun, 1994;
Weiss and Magleby, 1989
). Only
records or parts thereof with an overall steady-state P
o
should be used for kinetic
analysis. Files with stable baselines are exported as QuB-supported file formats (.ldt).
In QuB, the files are further examined and sections of data with spurious noise are
excluded using the preprocessing module (''Pre''). Current traces are then idealized
into noise-free, open, and closed transitions using the segmental k-means (SKM)
algorithm in the QuB suite. This uses a hidden Markov model (HMM) to decide
whether each excursion in the record should be classified as an open or closed state
based upon its amplitude (
Qin, 2004
)(
Fig. 4
B). The output at this stage is a categori-
zation of every transition into a switch between current amplitudes: a single closed
current amplitude (baseline noise) and one or several amplitudes of the open channel
(s). Where several evenly spaced current amplitudes are detected, it can be di
cult to
resolve whether they arise from openings of several channels or switches between
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