A NEW CAGED CA2-1, IS FAR MORE PHOTOSENSITIVE THAN NITROBENZYL-BASED CHELATORS(, AZID)

Background: Photolabile chelators that release Ca2+ upon illumination have been used extensively to dissect the role of this important secon d messenger in cellular processes such as muscle contraction and synap tic transmission, The caged calcium chelators that are presently avail able are often limited by their inadequate changes in Ca2+ affinity, s electivity for Ca2+ over Mg2+ and sensitivity to light, As these chela tors 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 Ca2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca2+ with a di ssociation constant (K-d) of similar to 230 nM, which changes to 120 m u M after photolysis with ultraviolet light (330-380 nm). Mg2+ binding is weak (8-9 mM K-d) before or after photolysis. Azid-1 photolyzes wi th unit quantum efficiency, making it 40-170-fold more sensitive to li ght than caged calciums used previously. The photolysis of azid-1 prob ably releases N-2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-withdrawing ability of the amidoxime ca tion reduces the chelator's Ca2+ affinity within at most 2 ms followin g a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in whic h Ca2+ photolytically released from azid-1 could replace the normal de polarization-induced Ca2+ transient in triggering synaptic plasticity, Conclusions: Azid-1 promises to be a useful tool for generating highl y 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 photo chemistry to a focal spot that is localized in three dimensions, Azide photolysis could be a useful and more photosensitive alternative to n itrobenzyl photochemistry.