Biology Reference
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technique for analysis of single-channel events. It has contributed enormously to
the understanding of gating and desensitization/inactivation of numerous ion
channels. However, most IP
3
R reside within intracellular membranes, where they
are inaccessible to conventional patch-clamp recording methods. Here, we describe
the application of nuclear patch-clamp methods to single-channel analyses of
native and recombinant IP
3
R.
I. Introduction
Inositol 1,4,5-trisphosphate receptors (IP
3
R) comprise a family of tetrameric
intracellular channels that mediate the release of Ca
2
þ
from the intracellular stores
of almost all animal cells (
Foskett et al., 2007; Taylor et al., 1999
). Three genes
encode homologous subunits of vertebrate IP
3
R, and a single gene encodes inver-
tebrate IP
3
R. The key structural determinants of IP
3
R activation, although pres-
ently poorly understood, are likely to be similar for all IP
3
R. Activation is initiated
by binding of IP
3
to a conserved IP
3
-binding core toward the N-terminal of each
subunit (
Bosanac et al., 2002
), conformational changes then pass via the
N-terminal suppressor domain (
Bosanac et al., 2005
) to the pore, which is formed
by transmembrane regions lying toward the C-terminus (
Boehning and Joseph,
2000; Foskett et al., 2007; Rossi et al., 2009; Taylor et al., 2004
). Most IP
3
R in most
cells are expressed within membranes of the endoplasmic reticulum (ER). Di
V
erent
IP
3
R subtypes may, however, di
er in their subcellular distributions (
Taylor et al.,
1999
) and in their modulation by various additional signals and associated pro-
teins (
Betzenhauser et al., 2008b; Choe and Ehrlich, 2006; Mackrill et al., 1997;
Patterson et al., 2004; Wojcikiewicz and Luo, 1998
). Resolving the roles of di
V
V
er-
ent IP
3
R subtypes in the genesis of the complex Ca
2
þ
signals that regulate cellular
activity is an important issue (
Futatsugi
et al., 2005; Miyakawa
et al., 1999;
Sugawara et al., 1997; Wang et al., 2001
).
Opening of the intrinsic pore of all IP
3
R requires binding of IP
3
and Ca
2
þ
(
Adkins and Taylor, 1999; Marchant and Taylor, 1997
). IP
3
R can, therefore,
both initiate the Ca
2
þ
signals evoked by receptors that stimulate IP
3
formation
and then regeneratively propagate them by Ca
2
þ
-induced Ca
2
þ
release. This dual
regulation of IP
3
R allows a hierarchical recruitment of Ca
2
þ
release events as the
stimulus intensity increases (
Bootman et al., 1997; Marchant and Parker, 2001
).
Single IP
3
R respond first, then several IP
3
R within a cluster open together to give
larger local events (''pu
s become more frequent, they ignite
regenerative Ca
2
þ
waves (
Bootman and Berridge, 1995; Marchant et al., 1999
).
This hierarchy of events allows Ca
2
þ
to function as a local or global messenger, a
feature that underlies its versatility (
Berridge et al., 2000
). A key point for the
present discussion is that local events involving very few IP
3
R underlie the Ca
2
þ
signals that regulate cellular activity. By contrast, for most ion channels, it is the
collective behavior of large numbers of channels, the macroscopic current, that
determines the physiological response, a change in membrane potential, or
V
s''), and as pu
V
