Biology Reference
In-Depth Information
Membrane-permeant
AMO
2
C
AMO
2
C
CO
2
AM
CO
2
AM
Indicator
Ca
2+
-insensitive
Ca
2+
-sensitive
−
O
2
C
CO
−
AMO
2
C
CO
2
AM
CO
2
AM
Esterases
−
O
2
C
CO
−
AMO
2
C
Indicator
Indicator
Trapped inside cell
Extracellular
Intracellular
Fig. 4
Schematic representation of how incubation with the acetoxymethyl (AM) ester results in
intracellular accumulation of a polycarboxylate indicator. The hydrophobic (lipophilic) AM ester
readily di
V
uses into the cell through the cell membrane. Abundant cellular esterases cleave the AM
ester groups to generate the Ca
2
þ
-sensitive form of the indicator which, being a polyanion, cannot
escape through the cell membrane and is, therefore, trapped inside the cell.
III. Loading Indicators into Cells
The common fluorescent indicators for Ca
2
þ
are polycarboxylate anions that
cannot cross lipid bilayer membranes and therefore are not cell-permeant. In the
negatively charged form, the indicators can be introduced into cells only by
microinjection or through transient cell permeabilization, procedures that require
some special equipment and skill.
5
By far the most convenient way of loading an
indicator into cells is incubating the cells in a dilute solution or dispersion of the
AM ester of the indicator. This process is represented schematically in
Fig. 4
. The
AM group is used to mask the negative charges on the carboxyl groups present in
the indicator molecule. The AM ester form of the indicator is uncharged and
hydrophobic. Consequently, it can pass through the cell membrane and enter the
cell interior. The carboxyl groups in the indicator, however, are essential to the
ability of the indicator molecule to sense Ca
2
þ
; therefore, the AM groups must be
removed once the AM ester has entered the cell. Because the AM group is labile to
enzymatic hydrolysis by esterases present in the cell, the AM esters are processed
intracellularly to liberate the Ca
2
þ
-sensitive polycarboxylate form which, being
multiply charged, becomes trapped inside the cell. Trapping of the polyanionic
form of the indicator allows cells to accumulate up to hundreds of micromolar of
5
A variety of techniques for loading membrane-impermeant species into cells is discussed by
McNeil
(1989, 2001)
.
