Biology Reference
In-Depth Information
CHAPTER 8
Nuclear Patch-Clamp Recording from
Inositol 1,4,5-Trisphosphate Receptors
Taufiq Rahman and Colin W. Taylor
Department of Pharmacology
Tennis Court Road, University of Cambridge
Cambridge, United Kingdom
Abstract
I. Introduction
II. Nuclear Patch-Clamp Recording
III. Choice of Cells for Analyses of IP
3
R
IV. Methods
A. Culture of DT40 Cells
B. Isolation of Nuclei
C. Solutions for Patch-Clamp Recording
D. Patch-Clamp Recording
E. Analysis of Single-Channel Records
V. Concluding Remarks
References
Abstract
Inositol 1,4,5-trisphosphate receptors (IP
3
R) are ubiquitous intracellular Ca
2
þ
channels. They are regulated by IP
3
and Ca
2
þ
and can thereby both initiate local
Ca
2
þ
release events and regeneratively propagate Ca
2
þ
signals evoked by receptors
that stimulate IP
3
formation. Local signaling by small numbers of IP
3
R underpins
the utility of IP
3
-evoked Ca
2
þ
signals as a ubiquitous signaling pathway. The
physiological impact of Ca
2
þ
release by very small numbers of IP
3
R underscores
the necessity to understand the behavior of IP
3
R at the single-channel level.
In addition, and in common with analyses of every other ion channel, single-
channel analyses have the potential to define the steps linking IP
3
binding to
channel opening. Patch-clamp recording, by resolving the openings and closings
of single channels with exquisite temporal resolution,
is the most powerful
