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fall in pH. If Mg 2 þ is also present, a secondary relaxation of [Ca 2 þ ] i follows because
of the slower displacement of Mg 2 þ from DM-nitrophen ( Ayer and Zucker, 1999;
Delaney and Zucker, 1990; Escobar et al., 1995, 1997 ). Moreover, if a steady UV
source is used to photolyze DM-nitrophen, rebinding continually lags release,
leading to a low (micromolar range) free [Ca 2 þ ] i while the illumination persists.
When the light is extinguished, the [Ca 2 þ ] i drops rapidly to a low level under control
of the remaining chelator ( Zucker, 1993 ). In the case of DM-nitrophen bound to
Mg 2 þ , achievement of equilibrium is somewhat slower (tens of milliseconds). Thus,
a reversible ''pulse'' of [Ca 2 þ ] i is generated, the amplitude of which depends on light
intensity and the duration of which is controlled by the length of the illumination.
This situation remains so until the remaining unphotolyzed cage becomes fully
saturated with Ca 2 þ , whereupon [Ca 2 þ ] i escapes from the control of the chelator,
imposing a practical limit on the product of [Ca 2 þ ] i and duration of about
0.75 m M s for DM-nitrophen. Similar kinetic considerations apply when Ca 2 þ is
passed by photolysis from a Ca 2 þ cage to another bu
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er such as BAPTA, EGTA,
HEEDTA, or DPTA. Judicious selection of bu
er ratios may be used to
shape this Ca 2 þ ''spike'' to match a hypothetical naturally occurring [Ca 2 þ ] i (t)
waveform and test its physiological consequences ( Bollmann and Sakmann,
2005 ). If this behavior is considered undesirable, it may be avoided by using only
fully Ca 2 þ -saturated DM-nitrophen, due to its extremely high Ca 2 þ -a
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ers and bu
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nity, for
which rebinding to unphotolyzed chelator is impossible. Thus, the kinetic complex-
ity of the nitrophen class of chelators can be turned to experimental advantage,
greatly magnifying the flexibility of experimental [Ca 2 þ ] i control.
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IV. Diazo Compounds
A. Chemical Properties
In some experiments, being able to lower the [Ca 2 þ ] i rapidly, rather than raise it,
is desirable. For this purpose, caged chelators were developed. Initial attempts
involved attachment of a variety of photosensitive protecting groups to mask one
of the carboxyl groups of BAPTA, thus reducing its Ca 2 þ a
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nity until restored by
photolysis. Such compounds displayed low quantum e
ciency ( Adams et al., 1989;
Ferenczi et al., 1989 ) and their development has not been pursued. A more suc-
cessful approach ( Adams et al., 1989 ) involved substituting one (diazo-2) or both
(diazo-4) of the aromatic rings of BAPTA with an electron-withdrawing diazoke-
tone that reduces Ca 2 þ a
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nity, much like the photoproducts of the nitr com-
pounds. Figure 4 shows the structures of the diazo series of chelators. Photolysis
converts the substituent to an electron-donating carboxymethyl group while
releasing a proton; the Ca 2 þ a
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nity of the photoproduct is thereby increased.
The reaction is illustrated in Fig. 5 .
Diazo-2 absorbs one photon with quantum e
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nity, in
433 m s, from 2.2 m M to 73 nM at 120-mM ionic strength (or to 150 nM at 250 mM
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ciency 0.03 to increase a
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