Biology Reference
In-Depth Information
calibration curve. Within this range, the higher the resistance, the lower the
detection limit for a given batch of MEs. At resistances higher than 300 G
O
, the
detection limit decreased sharply, probably because of the small tip diameter
(
Ammann, 1986
). Finally, although low resistance MEs tended to also have low
detection limits, they were not suitable for our experiments, because of the large tip
diameter that could seriously damage the cell membrane during impalement. By
contrast, we have found the height of the ligand column not to be a valuable
predictor of ME performance (although this height was always kept 50-250
m
m).
In our hands, an ME can be used a few minutes after ligand filling.
After equilibration in the control physiological solution (in our case, an HEPES-
based Tyrode, containing 2 mM Ca
2
þ
), ion-selective ME potential is adjusted to
0 mV. Our 2 mM Ca
2
þ
Tyrode gives a voltage reading corresponding to an
intracellular calibrating solution of pCa 2.6. Then, flow is switched to a solution
of high pCa (between 7.5 and 9). At 30
C (our experimental temperature), the
theoretical slope of the relationship between voltage and pCa is
30 mV/pCa, so
that, between pCa 2.6 and pCa 8, the theoretical voltage should be
162 mV.
However, as the electrode detection limit decreases at high pCa, a practical
compromise is often necessary for acceptability (e.g., readings more negative
than
150 mV). A Ca
2
þ
-selective ME meeting this criteria may then be calibrated
over a wider range of [Ca
2
þ
]. After the calibration is completed, the ME is moved
into the experimental chamber and equilibrated until stable. Conventional,
3 M KCl-filled MEs are pulled from the same glass and with the same character-
istics as the Ca
2
þ
electrodes, but are not silanized.
D. Application of Ca
2þ
-Selective MEs
1. Sharp Ca
2
þ
MEs for [Ca
2
þ
]
i
Measurement
We and others have had some, but limited success in making reliable [Ca
2
þ
]
i
measurements in cardiac myocytes with these electrodes (
Bers and Ellis, 1982;
Marban et al., 1980
), and a few other groups have had some luck with other cell
types such as skeletal muscle fibers (
Allen et al., 1992; Blatter and Blinks, 1991;
Lopez et al., 2000
), Aplysia neurones (
Gorman et al., 1984
), and photoreceptors
(
Levy and Fein, 1985
) using an earlier developed Ca
2
þ
ionophore (ETH 1001;
Sigma-Aldrich 21192) instead of ETH 129 in the cocktail. ETH 1001-based MEs
generally do not make electrodes that have quite as low a detection limit as ETH
129 (
Schefer et al., 1986
), but for some reason ETH1001 seems to be of greater
practical utility for intracellular Ca
2
þ
MEs. We have done some preliminary
cardiac muscle experiments
that are consistent with this notion (resting
pCa
10 determinations). However, with the excellent
fluorescent Ca
2
þ
indicators available now that are easy to use, one would need a
compelling reason to tackle this challenging electrophysiological approach for
[Ca
2
þ
]
i
and the references above should then help.
¼
6.34
0.15; mean
SD; n
¼
