Biology Reference
In-Depth Information
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
Adkins, C. E., and Taylor, C. W. (1999). Lateral inhibition of inositol 1, 4, 5-trisphosphate receptors by
cytosolic Ca 2 þ . Curr. Biol. 9, 1115-1118.
Baba, T. W., Giroir, B. P., and Humphries, E. H. (1985). Cell lines derived from avian lymphomas
exhibit two distinct phenotypes. Virology 144,
139-151.
Search WWH ::




Custom Search