Biology Reference
In-Depth Information
transcellular ion flux, for example. The distinction highlights the particular impor-
tance of single-channel recording in the analysis of IP
3
R. As with all such analyses,
they provide the highest resolution insight into the opening and closing of single
channels and can thereby reveal details of gating mechanisms (
Colquhoun, 2007;
Sivilotti, 2010
). But for IP
3
R and other Ca
2
þ
channels too, openings of individual
channels are the physiologically significant behavior.
The patch-clamp technique, developed originally by
Neher and Sakmann (1976)
with subsequent improvements (
Hamill et al., 1981
), is the most powerful means of
studying the behavior of ion channels in their native environment. It involves
recording currents passing through an electrically isolated, small area (''patch'')
of biological membrane in response to an applied voltage or ionic gradient
(
Fig. 1
A and B). Isolation of the patch is achieved by pressing a polished glass
pipette tip of
1
m
m diameter (containing electrolyte solution) against the cell-
surface and applying gentle suction to form a very high-resistance ''giga-Ohm''
(G
O
) seal (
Hamill et al., 1981
). The tight seal is crucial because it isolates the patch
both electrically and physically, so reducing background noise and allowing single-
channel events to be resolved (
Hamill et al., 1981
). Because of the electrical
isolation and low resistance of the patch-pipette relative to the membrane, a
patch can be voltage-clamped by simply applying a potential to the pipette.
These patch-clamp recordings allow the openings and closings of individual chan-
nels to be resolved with submillisecond temporal resolution under optimal condi-
tions (
Fig. 1
C). The amplitudes of these tiny currents and their dependence on
applied potential and ion concentrations allow the ion selectivity and rates of ion
permeation to be determined. Lurking within the pattern of stochastic openings
and closings is the information from which the sequence of events that leads to
channel gating and desensitization/inactivation can be reconstructed. Comprehen-
sive descriptions of the patch-clamp technique are available from the original
articles (
Hamill et al., 1981; Neher and Sakmann, 1976
) and subsequent reviews
(
Ogden, 1994; Sakmann and Neher, 1995
). However, most IP
3
R are expressed in
membranes of the ER, where they are inaccessible to conventional patch-clamp
techniques. Alternative approaches are therefore needed.
II. Nuclear Patch-Clamp Recording
It is impracticable, despite one heroic success recording from IP
3
R within the
ER of an intact cell (
Jonas et al., 1997
), to use patch-clamp techniques routinely to
record the behavior of single channels within the membranes of intracellular
organelles in situ. A more promising approach for single-channel recordings
in situ is the ''optical patch-clamp,'' where high-resolution optical microscopy in
combination with fluorescent Ca
2
þ
indicators is used to measure the Ca
2
þ
signals
evoked by opening of single or small clusters of IP
3
R(
Demuro and Parker, 2007;
Smith and Parker, 2009
). Presently, however, these optical methods can be used
only to measure fluxes through Ca
2
þ
channels, and they lack the temporal
