Biology Reference
In-Depth Information
the K
d
for EGTA, BAPTA, and oxalate in bu
er solutions (
Bers, 1982; Harrison
and Bers, 1987, 1989; Hove-Madsen and Bers, 1993a
). More comprehensive pro-
grams to calculate the amount of Ca
2
þ
and Ca
2
þ
bu
V
er needed to prepare solu-
tions have been developed (e.g., MaxChelator by C. Patton, Marine Biology
Institute, Monterey, CA;
http://maxchelator.stanford.edu/downloads.htm; cf
.
Chapter 1).
We have also used the minielectrodes to characterize the binding of Ca
2
þ
to
indo-1 in vitro and in cell suspensions, in order to calibrate the indo-1 and furaptra
signals when used in cell suspensions (
Hove-Madsen and Bers, 1992; Hove-
Madsen et al., 1998; Shannon and Bers, 1997
). In agreement with previous studies
of fura-2 (
Konishi et al., 1988
) we found that indo-1 binds extensively to cellular
proteins and causes a
V
fourfold increase in the K
d
for Ca
2
þ
-indo-1 in permeabi-
lized myocytes.
The minielectrodes can also be used to titrate the passive Ca
2
þ
binding sites in
permeabilized myocytes where the cellular Ca
2
þ
uptake and release process are
inhibited. Using the same titration method, we have measured total Ca
2
þ
uptake in
the SR in permeabilized myocytes, by inhibiting Ca
2
þ
uptake in the mitochondria
and release of Ca
2
þ
through the SR Ca
2
þ
release channels (
Hove-Madsen and
Bers, 1993a
).
Finally, we have used the minielectrodes together with indo-1 for online mea-
surements of the Ca
2
þ
uptake rate in the SR in permeabilized ventricular myocytes
(
Hove-Madsen and Bers, 1993b
) and to examine the e
ects of phospholamban
phosphorylation and temperature on the uptake rate (
Hove-Madsen et al., 1998;
Mattiazzi et al., 1994
), and we have determined the inhibition of Ca
2
þ
uptake by
thapsigargin in order to measure the number of SR Ca
2
þ
pump sites (
Hove-
Madsen and Bers, 1993b
). In experiments measuring Ca
2
þ
uptake rates caution
should, however, be taken when using minielectrode because of the above men-
tioned possibilities of inhomogeneities in Ca
2
þ
in cell suspensions and slowing of
the electrode response.
V
C. Preparation of Ca
2þ
-Selective MEs
1. Glass Tubing Preparation
We have used nonfilamented capillaries (150
m
m outer diameter, 15 cm long,
from Clark Electromedical Instruments, UK or World Precision Instruments,
USA). The glass is cut in the middle with a diamond pen or glass scorer (with
care to avoid deposition of dirt). Both ends of the capillaries (now 7.5 cm long) are
lightly fire-polished; a whole batch can be prepared and kept in a small glass
beaker, preferentially in a dust-proof container. Cleaning of the micropipettes
prior to pulling has been a matter of debate, but as 99.8% of the glass after pulling
is newly exposed (
Deyhimi and Coles, 1982
), we do not find such procedures to be
necessary.
