Biology Reference
In-Depth Information
Abstract
Ca
2
þ
signaling in the extra- and intracellular domains is linked to Ca
2
þ
transport across the plasma membrane. Noninvasive monitoring of these result-
ing extracellular Ca
2
þ
gradients with self-referencing of Ca
2
þ
-selective micro-
electrodes is used for studying Ca
2
þ
signaling across Kingdoms. The quantitated
Ca
2
þ
flux enables comparison with changes to intracellular [Ca
2
þ
]measuredwith
other methods and determination of Ca
2
þ
transport stoichiometry. Here, we
review the construction of Ca
2
þ
-selective microelectrodes, their physical charac-
teristics, and their use in self-referencing mode to calculate Ca
2
þ
flux. We also
discuss potential complications when using them to measure Ca
2
þ
gradients near
the boundary layers of single cells and tissues.
I. Introduction
Regulation of resting [Ca
2
þ
]
i
and the control of spatial and temporal dynam-
ics during Ca
2
þ
signaling require coordinated transport between membrane-
separated compartments, giving rise to Ca
2
þ
fluxes across organelles and the
plasma membrane. Movement of Ca
2
þ
across the plasma membrane via trans-
porters, exchangers, or channels gives rise to minute gradients of [Ca
2
þ
]inthe
extracellular boundary layer that reflect changes in [Ca
2
þ
]
i
. The near real-time
extraction of these gradients requires a detection method that is not disturbing
to the local chemical environment, functions over a wide dynamic range, and
possesses high sensitivity, selectivity, and spatial resolution. For these reasons
extracellular Ca
2
þ
gradients have been monitored with self-referencing of Ca
2
þ
-
selective microelectrodes (CaSMs), enabling noninvasive characterization of
Ca
2
þ
transport and signaling events. Unlike most fluorescent or luminescent
indicators, CaSMs were originally developed for measuring both intracellular
and extracellular [Ca
2
þ
] (listed in
Lanter et al., 1982
). Measurement of minute
Ca
2
þ
gradients on the outside of cells was limited by electrical drift in the
system. For this reason, a modulation technique was introduced (
K¨htreiber
and Ja
e, 1990
) that enabled reduction of drift and provided a simple means
for calculating Ca
2
þ
flux. The method was later coined ''self-referencing'' and
has been extended to other ion-selective microelectrodes and amperometric
microelectrodes enabling characterization of fluxes of many di
V
erent analytes
(
Messerli et al., 2006; Smith et al., 2007
).MeasurementofCa
2
þ
fluxes with self-
referencing has enabled direct comparison of Ca
2
þ
fluxes measured with other
techniques including radioactive tracers, fluorescent and luminescent ion indi-
cators, and voltage clamp. We will first discuss the construction and general
properties of CaSMs before discussing their use with the self-referencing
approach.
V
