Biology Reference
In-Depth Information
100 nMCa
2
þ
in the backfilling solution generates an influx of Ca
2
þ
into the CaSM
tip, depleting the local [Ca
2
þ
] in the bath and giving rise to an artificial Ca
2
þ
influx
into the glass bead. Again this can be detected in the 50
m
MCa
2
þ
solution but not
the 2 mM Ca
2
þ
containing bath solution. The artifact can be reduced by matching
the backfilling [Ca
2
þ
] with the [Ca
2
þ
] in the bath. Other methods for eliminating
Ca
2
þ
flux across the tip of the CaSM include current clamping (
Lindner et al.,
1999; Pergel et al., 2001
), or using the solid contact ion-selective electrode design
(
Lindner and Gyurcs´nyi, 2009
).
V. C a
2
þ
Flux Measurements
Extracellular Ca
2
þ
flux measurements have been performed on a number of
di
erent systems some of which are listed in
Table IV
, ranging from animal
neurons and muscle to tip growing root hairs, pollen tubes, and fungi. Measured
Ca
2
þ
fluxes are relatively small ranging between 0.1 and 10 pmol cm
2
s
1
encour-
aging measurements from cells in reduced background [Ca
2
þ
]
V
0.1 mM. The limit
of flux sensitivity for a typical self-referencing CaSM with
10
m
V near real-time
variation performed in 1 mM bath [Ca
2
þ
] is about
6.3 pmol cm
2
s
1
, an order
of magnitude higher than in 0.1 mM bath [Ca
2
þ
]. Considering the large trans-
plasma membrane electrochemical driving force on Ca
2
þ
, reduction of extra-
cellular [Ca
2
þ
] by an order of magnitude did not cause noticeable problems
for the di
erent preparations, at least over the few hour period during
which measurements were acquired as noted by multiple authors listed in
Table IV
.
While e
V
ux of Ca
2
þ
in cells at rest is expected to be relatively small, the
measured influx of Ca
2
þ
, presumably through channels, is also relatively small.
Active single 0.5 pS Ca
2
þ
channels at a density of 1
m
m
2
should give rise to a Ca
2
þ
influx of about 47 pmol cm
2
s
1
. Although as noted by
Hille (2001)
voltage-gated
Ca
2
þ
channels exist at low density and low open probabilities (
Z
0.1) even with
strong depolarizing potentials indicating that low channel density and activity is
su
<
cient to account for measured changes in [Ca
2
þ
]
i
. The channel density and
activity used above may be overestimates of actual Ca
2
þ
channel density. Also,
weak influx may also be a result of the Ca
2
þ
amplification cascades that exist to
release Ca
2
þ
from intracellular stores after influx through the plasma membrane.
Additional directions for the use of self-referencing with CaSMs include the
study of electroneutral Ca
2
þ
transporters/exchangers and extracellular Ca
2
þ
sig-
naling (
Breitwieser, 2008
). Ca
2
þ
selective microelectrodes have been instrumental
in providing the sensitivity for defining the complex transport of the Na
þ
/Ca
2
þ
exchanger (
Kang and Hilgemann, 2004
) and the P-type plasma membrane Ca
2
þ
pump (PMCA) in neurons (
Thomas, 2009
). With self-referencing of ion-selective
microelectrodes, transport stoichiometries could be determined noninvasively
from the outside of intact cells.
Y
