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Rahman et al.,2009 ), human B lymphoblasts ( Cheung et al.,2008 ), cerebellar
Purkinje cells ( Marchenko et al.,2005 ), and embryonic cortical neurons and fibro-
blasts ( Cheung et al.,2008 ).
The utility of nuclear patch-clamp recording derives from the fact that the outer
nuclear envelope is continuous with the ER membrane ( Dingwall and Laskey,
1992 )( Fig. 2 A). Channels that are normally expressed within ER membranes can,
therefore, pass into the outer nuclear envelope, allowing their activity to be
recorded from patches of nuclear membrane in a near-physiological setting
( Fig. 2 ). Abundant nuclear pore complexes, each with a large central conduit
linking cytoplasm and nucleoplasm ( Mazzanti et al., 2001 ), might have been
expected to compromise formation of the tight seals required for patch-clamp
recording or at least pollute recordings from conventional channels with lesser
conductances. In practice nuclear pores appear not to cause problems. It seems
unlikely, though it remains possible, that this results from patching onto ER
overlying the nuclear envelope, rather than the envelope itself. It is perhaps more
likely that patches that include nuclear pores are rejected because they fail to form
giga-Ohm seals, or the high-K รพ medium used for nuclear patch-clamp recording
favors closure of nuclear pores ( Bustamante and Varanda, 1998 ).
III. Choice of Cells for Analyses of IP 3 R
Almost all animal cells express IP 3 R, most express more than one of the three
vertebrate gene products, and substantial alternative splicing and posttranslational
modifications add further to the diversity of subunits from which IP 3 R are assem-
bled ( Foskett et al., 2007 ). Assembly of these subunits into homo- and heterote-
trameric structures increases the diversity of functional IP 3 R enormously ( Joseph
et al., 1995, 2000; Wojcikiewicz and He, 1995 ). Despite this complexity, there have
been many valuable studies of the single-channel behavior of native IP 3 R in, for
example, Xenopus oocytes, nuclei from oocytes, insect Sf9 cells, and cerebellar
Purkinje neurons, and of native IP 3 R reconstituted into lipid bilayers ( Section II ).
But the limitations of such studies are obvious when it to comes to exploring the
structural basis of IP 3 R activation. This demands a more homogenous population
of IP 3 R with a defined structure and ideally expressed in a native membrane.
At present, only one expression system provides the ''null background'' that allows
these demanding criteria to be satisfied: DT40 cells ( Fig. 3 ).
DT40 cells originate from an avian leukosis virus-transformed bursal B cell
( Baba et al., 1985 ). The uniquely valuable feature of these cells is the unusually
high frequency with which they integrate targeted DNA constructs into their
genome ( Buerstedde and Takeda, 1991 ). This feature, together with the shorter
introns of avian genes, allows targeted disruption of specific genes and has ensured
widespread use of DT40 cells for ''gene-knockouts.'' In a monumental e
ort,
Kurosaki and his colleagues used targeted gene disruption to inactivate both
copies of all three IP 3 R genes in DT40 cells and thereby to generate the first cell
V
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