Biology Reference
In-Depth Information
pClamp 9.2), which uses the generalized Henderson equation (
Barry, 1994
). The
calculated LJP is then subtracted from the observed reversal potential (E
rev
)
obtained from the current-voltage (I-V) plot. We return in
Section IV.E
, after
considering analysis of the raw traces, to describe how E
rev
allows the relative
permeability of IP
3
Rtodi
V
erent cations to be calculated.
In both the on-nucleus and excised patch configurations described above, the
cytoplasmic surface of the IP
3
R lies within the patch-pipette (
Fig. 2
C); it is, therefore,
di
cult to change the IP
3
concentration once the giga-Ohm seal has formed. It is
possible, though di
Y
cult, to perfuse a patch-pipette and thereby to vary the composi-
tion of the ''cytosolic'' medium while recording channel activity (
Hering et al.,1987;
Maathuis et al.,1997
), but this technique has not yet been applied to IP
3
R. Other
options include the cytoplasm-out configuration of nuclear patch-clamp recording
(
Fig. 2
C), which has been successfully applied to analyses of IP
3
R in Sf9 cells (
Mak
et al.,2007
). Alternatively, flash-photolysis of caged-IP
3
within the patch-pipette in
either the on-nucleus or excised nuclear patch configuration can be used rapidly to
increase the IP
3
concentration bathing the cytosolic surface of the IP
3
Roncethe
recording is underway (
Rahman et al.,2009
). For these flash-photolysis experiments,
pipettes are prepared from thin-walled, nonfilamented borosilicate glass capillaries
(Harvard Instruments) and PS includes
Y
d
-myo-inositol 1,4,5-trisphosphate, (4,5)-1-
(2-nitrophenyl) ethyl ester (caged-IP
3
,
100
m
M, Calbiochem). After recording for
30-60 s, IP
3
can thenbe released intoPSbyphotolysis of caged-IP
3
using a single high-
intensity flash (1 ms) from a Xe-flash lamp (XF-10, Hi-Tech Scientific; 240 J with the
capacitor charged to 385 V) passed through a filter (300-350 nM) (
Walker et al.,
1987
). A problemwith this approach is the di
culty in assessing the concentration of
IP
3
to which the IP
3
R are exposed after flash-photolysis of caged-IP
3
.
Y
E. Analysis of Single-Channel Records
Two sorts of information can be extracted from single-channel records: the
properties of the open channel (its ability to conduct di
erent ions); and the
sequence of stable states through which the channel passes as it moves between
closed, open, and desensitized conditions. Here, we provide only a brief introduc-
tory summary of the methods used to extract this information from the openings
and closings of channels resolved by patch-clamp recording. The reader interested
in more rigorous and detailed descriptions is advised to begin with two excellent
topics (
Ogden, 1994; Sakmann and Neher, 1995
).
Several software packages are available for analysis of electrophysiological
records. These include ClampFit, which includes the pClamp suite (Molecular
Devices), and DC-soft, which includes SCAN, EKDIST, and HJCFIT (
http://
V
alo.edu
), Pulse/
Patchmaster (HEKA Elecktronik), Tac (Bruxton Inc.), and the Strathclyde Electro-
physiology Software (
http://spider.science.strath.ac.uk/sipbs/software_ses.htm
).
We use ClampFit and QuB for analyzing records (
Dellis
V
et al., 2006; Rahman
et al.,2009
).
