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composition (
Taylor et al., 2009b; Tovey et al., 2006
). Nuclear patch-clamp
recording of DT40 cells heterologously expressing mammalian IP
3
R, therefore,
allows single-channel recording with its exquisite temporal resolution to be com-
bined with opportunities to manipulate systematically the structure of the
expressed IP
3
R. The stability of these patch-clamp recordings in a native mem-
brane and the opportunity to apply them in various configurations (
Fig. 2
)a
ord
valuable opportunities to examine the behavior of small numbers of IP
3
R directly
(
Rahman et al., 2009
) and as a means to address the mechanisms underlying
IP
3
R activation (
Rossi et al., 2009
).
In the short period during which nuclear patch-clamp analyses have been
applied to IP
3
R, they have succeeded in confirming that IP
3
R are large conduc-
tance, relatively nonselective cation channels, and revealed the durations of the
channel openings and closing (
Dellis et al., 2006; Foskett et al., 2007; Rahman
et al., 2009
)(
Figs. 4-6
). Together, these insights allow estimates of the likely Ca
2
þ
fluxes through individual IP
3
R for comparison with optical measurements of the
elementary Ca
2
þ
signals evoked by IP
3
in situ (
Shuai et al., 2007, 2008
). Combining
site-directed mutagenesis with nuclear patch-clamp recording has provided direct
evidence that the pore of IP
3
R is formed by residues within the ''P-loop'' linking
the final pair of transmembrane domains of each IP
3
R subunit (
Boehning et al.,
2001b; Dellis et al., 2006, 2008; Schug et al., 2008
). The e
V
ects of a novel family of
synthetic partial agonists on normal and mutant IP
3
R analyzed by nuclear patch-
clamp recording have shed light on the first stages of IP
3
R activation by showing
that the initial conformation changes evoked by IP
3
binding to the IP
3
-binding
core pass onward toward the pore entirely via the N-terminal suppressor domain
(
Rossi et al., 2009
). Similar analyses have revealed the means, whereby ATP
(
Betzenhauser et al., 2008b, 2009b
), cyclic AMP-dependent protein kinase
(
Betzenhauser et al., 2009a
), cyclic AMP (
Tovey et al., 2010
), and various accesso-
ry proteins (
Cheung et al., 2008, 2010; Li et al., 2007
) modulate IP
3
R behavior.
Future application of the nuclear patch-clamp technique to IP
3
R is certain to add
further to our understanding of the stochastic behavior of single and clustered
IP
3
R and to resolving the structural basis of IP
3
R activation.
V
Acknowledgments
This work was supported by grants from the Wellcome Trust, and the Biotechnology and Biological
Sciences Research Council (UK). T. R. is a Drapers' Company Research Fellow at Pembroke College,
Cambridge.
References
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