Nuclear Patch-Clamp Recording from Inositol 1,4,5-Trisphosphate Receptors - Methods in Cell Biology - page 200

resolution of conventional patch-clamp recording. Measuring the electrical activity
of intracellular channels, therefore, presently relies upon redirecting channels to the
plasma membrane, where they then become accessible to conventional patch-clamp
techniques ( Xu et al.,2007 ); reconstituting the channel into an artificial membrane;
or isolating organelles that express the channel and adapting the patch-clamp
technique to record from these membranes. The latter has been used, for example,
to resolve the behavior of the mitochondrial Ca 2 þ uniporter from mitochondria
stripped of their outer membrane (''mitoplasts'') ( Kirichok et al.,2004 ) and for
single-channel recordings of the endolysosomal protein, TRPML1, from artificially
enlarged lysosomes ( Dong et al.,2008 ). All three methods have been used to record
single-channel behavior of IP 3 R.
We observed that DT40 cells express very small numbers of functional
IP 3 R within the plasma membrane ( Dellis et al., 2006 ) and because DT40 cells
lacking native IP 3 R are available ( Section III ), conventional whole-cell patch-
clamp recording has been used by us ( Dellis et al., 2008 ) and others
( Betzenhauser et al., 2008b, 2009a ) to examine the behavior of recombinant and
mutant IP 3 R. Typical recordings from IP 3 R in the plasma membrane of DT40 cells
are shown in Fig. 1 C. A limitation of this approach is that excised patch-clamp
recording, where the ''intracellular'' composition can be precisely controlled, is
impracticable (because plasma membrane IP 3 R are too scarce), and with whole-
cell recording, it is di cult to define reliably the exact concentration of IP 3 bathing

the IP 3 R( Dellis et al., 2006 ). Detailed descriptions of the methods used for whole-
cell recording of IP 3 R expressed in the plasma membrane of DT40 cells have been
published ( Dellis et al., 2006; Taylor et al., 2009b ). We prefer nuclear patch-
clamping (see below) to whole-cell recording because the nuclear envelope is
continuous with the ER ( Fig. 2 A), wherein reside most IP 3 R, and it is practicable
to work with excised patches that provide better control of media bathing both
sides of the membrane.
The first electrical recordings from IP 3 R were made by incorporating native or
purified IP 3 R into artificial lipid bilayers ( Bezprozvanny et al., 1991; Ehrlich and
Watras, 1988; Maeda et al., 1991; Mayrleitner et al., 1991 ). As with all reconsti-
tuted systems, the lipid composition of the bilayer and the steps involved in
isolating, purifying, and reconstituting IP 3 R into the bilayer may a

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ect normal
function of the channel, not least its regulation by accessory proteins ( Boehning
et al., 2001a; Foskett et al., 2007; Patterson et al., 2004 ). Anecdotally, and it
equates with our experience, it seems to be more di

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cult to obtain bilayer record-
ings from IP 3 R than from its close relatives, the ryanodine receptors ( Williams,
1995 ).
Many of the problems with bilayer recording are resolved by using nuclear patch-
clamp recording ( Fig. 2 ). This technique was first introduced in the early 1990s
( Matzke et al., 1990; Mazzanti et al., 1990, 2001; Tabares et al.,1991 ) and subse-
quently, applied by the laboratories of Clapham ( Stehno-Bittel et al.,1995 )and
Foskett ( Mak and Foskett, 1994 ) to record single-channel events from native
IP 3 R in nuclei from Xenopus oocytes. It has, subsequently, been successfully applied

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