Biology Reference
In-Depth Information
to the micromolar range such as fura-2, indo-1, fluo-4, and many others. In this
chapter, we emphasize the utility of Ca
2
þ
-selective electrodes and show that their
use is complementary to use of fluorescent indicators; indeed, each method has
advantages and disadvantages. We first describe the preparation and application
of Ca
2
þ
-selective minielectrodes based on the Ca
2
þ
ligand ETH 129 (Schefer
et al., 1986) that have a larger dynamic range and faster response time than most
commercially available calcium electrodes. The second part of the chapter is
dedicated to ETH 129-based Ca
2
þ
-selective microelectrodes (MEs), and their
application in the determination of [Ca
2
þ
]
i
in cardiac cells. Since numerous
reviews and topics have been dedicated to the theoretical aspects of ion-selective
ME principles and technology, this chapter is not intended for investigators who
have no experience with MEs.
I. Introduction
A. Main Characteristics of Ca
2þ
-Selective Electrodes
The key advantage of the Ca
2
þ
-selective electrodes is the wide dynamic range
of their response (e.g., from pCa 9 to 1), as compared, for example, to
fluorescent and metallochromic Ca
2
þ
indicators that typically have a dynamic
range of four or less pCa units (
Fig. 1
). There has been developed a plethora of
useful fluorescent calcium probes with calcium sensitivities varying from the
nanomolar to the micromolar range such as fura-2, indo-1, fura red, fluo-4,
furaptra, fluo-5N, and others (
Grynkiewicz et al., 1985; Harkins et al., 1993;
Lipp et al., 1996; Picht et al., 2006; Shannon and Bers, 1997
). These are widely
used and are extremely important tools for study of Ca
2
þ
, but Ca
2
þ
-selective
electrodes are a valuable complementary tool. For more basic reference to
electrode technology and electrophysiology, we suggest monographs by
Ammann (1986), Purves (1981), and Thomas (1982)
. An electronic introduc-
tion to ion-selective electrodes can be found at
www.nico2000.net/Book/
Their response is based on a semiempirical equation (Nicolski-Eisenman equa-
tion) derived from the Nernst equation:
xy
a
zx=zy
pot
E
x
¼ E
o
þ RT=Z
x
F
ln
ða
x
þ K
Þ
ð
1
Þ
y
where E
x
is the ion-selective electrode potential, E
o
is a constant, R, T, Z,andF
have their usual meaning, a
x
is the activity of the ion that is measured (activity
(a) is related to concentration (C) by the relation: a
¼g
C where
g
is the activity
coe
pot
cient. This expression is strictly
valid for activities only, but if the activity coe
Y
cient) and K
xy
is the selectivity coe
Y
cients do not change, they can
be used with free concentrations too. This is often for convenience, since
solutions and chemical equilibria are more often described in these concentra-
tions terms. So, if x is Ca
2
þ
and y is Na
þ
(the most common interfering cation
Y
