Biology Reference
In-Depth Information
CO
−
−
O
2
C
−
O
2
C
CO
−
−
O
2
C
CO
−
N
N
N
N
N
N
N
N
N
O
O
O
H
3
C
TPEN
BAPTA
Half-BAPTA
Fig. 10
Structures of TPEN, BAPTA, and half-BAPTA.
millimolar levels of Ca
2
þ
. That TPEN is membrane-permeant means it can be
applied without using any special procedures. Dry DMSO can be used to prepare
stock solutions of TPEN. Typically, TPEN is used in aqueous medium at a
concentration of 10
6
-10
5
M.
BAPTA loading through the AM ester is very e
cient; high concentrations of
BAPTA may be accumulated intracellularly. Thus, ascertaining that observed
inhibitory e
Y
ects are not the result of cytotoxicity arising from the presence of
high concentrations of a foreign organic anion may be important. In this case, the
control reagent is N-(o-methoxyphenyl)iminodiacetic acid,
19
sometimes referred
to as ''half-BAPTA.'' As can be seen from
Fig. 10
, half-BAPTA is essentially
chemically identical to BAPTA except that the molecule is only half of BAPTA.
Because the full tetracarboxylate structure of BAPTA is crucial for Ca
2
þ
binding,
half-BAPTA, lacking such a structure, shows only very weak a
V
nity for Ca
2
þ
Y
(K
d
3 mM; J.P.Y. Kao, unpublished results). Half-BAPTA is thus expected to
mimic BAPTA in all chemical respects except for the ability to bu
er Ca
2
þ
at
physiological concentrations. The AM ester of half-BAPTA is sporadically
available from commercial vendors. Cell loading via the AM ester can be done
as described previously for other AM esters.
20
V
V. Conversion of Indicator Fluorescence Signal into
Values of [Ca
2
þ
]
Although raw fluorescence signals from intracellularly trapped Ca
2
þ
indicators
can be informative in a qualitative way, one still must perform some calibration
before even semiquantitative estimates of [Ca
2
þ
]
i
can be made. Basic principles of,
19
A trivial name is anisidine-N,N-diacetic acid.
20
Because it is processed by esterases to generate only the Ca
2
þ
-insensitive half-BAPTA and yet it is
processed intracellular in the same way that all AM esters are, half-BAPTA AM can also be used as a
control for possible artifacts from AM ester hydrolysis.
