Photolabile chelators that release Ca2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle contraction and ...synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca2+ affinity, selectivity for Ca2+ over Mg2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties.
Azid-1 is a novel caged calcium in which a fluorescent Ca2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca2+ with a dissociation constant (Kd) of approximately 230 nM, which changes to 120 microM after photolysis with ultraviolet light (330-380 nm). Mg2+ binding is weak (8-9 mM Kd) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40-170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-withdrawing ability of the amidoxime cation reduces the chelator's Ca2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca2+ transient in triggering synaptic plasticity.
Azid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca2+ in studies of the many Ca2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.
Background: Photolabile chelators that release Ca
2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle ...contraction and synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca
2+ affinity, selectivity for Ca
2+ over Mg
2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties.
Results: Azid-1 is a novel caged calcium in which a fluorescent Ca
2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca
2+ with a dissociation constant (K
d) of ∼230 nM, which changes to 120 μM after photolysis with ultraviolet light (330–380 nm). Mg
2+ binding is weak (8–9 mM K
d) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40–170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N
2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-with-drawing ability of the amidoxime cation reduces the chelator's Ca
2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca
2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca
2+ transient in triggering synaptic plasticity.
Conclusions: Azid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca
2+ in studies of the many Ca
2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.
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