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sensitivities to uniform postsynaptic [Ca 2 þ ] i elevation by NP-EGTA photolysis vs.
the volume-average of the highly nonuniform [Ca 2 þ ] i elevation on opening voltage-
sensitive Ca 2 þ channels by depolarizations ( Wang and Zucker, 2001 ), implying
that the enzymatic targets of postsynaptic Ca 2 þ entering through Ca 2 þ channels in
activating DSI are not tightly colocalized with the channels—a situation exactly
opposite of the case for Ca 2 þ activation of classical transmitter release.
Long-lasting synaptic regulation also occurs at developing neuromuscular junc-
tions, where repeated activation of one of two motor neuron inputs results in a
postsynaptic Ca 2 þ -dependent compensatory or homeostatic reduction in presyn-
aptic transmitter release to action potentials at terminals facing the activated
receptors. Using focal DM-nitrophen or nitr-5 photolysis to mimic the localized
postsynaptic [Ca 2 þ ] i elevation seen to accompany the stimulus normally used to
induce this selective persistent depression, we were able to induce a similar syn-
apse-specific modification ( Cash et al., 1996a ). Subsequently, synapses made by the
modified motor neuron onto other muscle fibers also became depressed by the
spread of an unidentified presynaptic intracellular signal ( Cash et al., 1996b ).
D. Other Applications
The tight regulation of cytoplasmic [Ca 2 þ ] i is essential for ensuring that Ca 2 þ can
act reliably and e
ciently as a localized second messenger of a huge variety of
cellular processes. Endogenous bu
Y
ers play a defining role in this process, and an
appreciation of the functions of these bu
V
nities,
binding kinetics, mobility, and localization) is crucial to our understanding how
Ca 2 þ performs its central cellular functions. Use of photosensitive Ca 2 þ chelators
has become an important tool in the estimation of cytoplasmic bu
V
ers and their characteristics (a
Y
V
er characteristics,
V
and much e
ort has gone into developing procedures and protocols for defining
them with some precision. Some of the best examples of this sort of analysis come
from the laboratories of Stephen Bolsover, Istvan Mody and Julio Vergara, and
Erwin Neher, whose papers should be consulted for the analytical details ( Faas et al.,
2007; Fleet et al.,1998;N¨gerl et al., 2000; Naraghi et al., 1998; Xu et al.,1997 ).
In addition to these major areas of application of caged Ca 2 þ chelators, this
method of [Ca 2 þ ] i manipulation has been used to address an increasingly diverse
range of biological problems. Nitr and diazo compounds were inserted by AM
loading into fibroblasts that were activated by mitogenic stimulation to produce
[Ca 2 þ ] i oscillations monitored using f1uo-3 ( Harootunian et al., 1988 ). Photore-
lease of Ca 2 þ from nitr-5 enhanced and accelerated the oscillations, whereas
release of caged chelator by photolysis of diazo-2 inhibited them. Nitr-7 photolysis
caused not only an immediate rise in [Ca 2 þ ] i liberated from the photolyzed chela-
tor, but also elicited a later rise in [Ca 2 þ ] i ( Harootunian et al., 1991 ). This e
ect was
shown, pharmacologically, to be caused by IP 3 -sensitive stores, suggesting that an
interaction between [Ca 2 þ ] i and these stores underlies the [Ca 2 þ ] i oscillations.
Photorelease of Ca 2 þ from DM-nitrophen has been used to study the binding
kinetics of Ca 2 þ to the Ca 2 þ -ATPase of sarcoplasmic reticulum vesicles ( DeLong
V
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