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J. Biol. Chem., Vol. 282, Issue 35, 25517-25526, August 31, 2007

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Synapse Specificity of Calcium Release Probed by Chemical Two-photon Uncaging of Inositol 1,4,5-Trisphosphate^*

Dmitry V. Sarkisov, Shari E. Gelber^¶, Jeffery W. Walker^||, and Samuel S.-H. Wang^**1

From the Departments of Physics and ^**Molecular Biology and Program in Neuroscience, Princeton University, Princeton, New Jersey 08544, ^¶Division of Maternal and Fetal Medicine, Cornell Weill Medical College, New York, New York 10021, and ^||Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706

Biological messengers can be "caged" by adding a single photosensitive group that can be photolyzed by a light flash to achieve spatially and temporally precise biochemical control. Here we report that photolysis of a double-caged form of the second messenger inositol 1,4,5-trisphosphate (IP₃) triggers focal calcium release in Purkinje cell somata, dendrites, and spines as measured by two-photon microscopy. In calbindin knock-out Purkinje cells, peak calcium increased with flash energy with higher cooperativity for double-caged IP₃ than for conventional single-caged IP₃, consistent with a chemical two-photon effect. Spine photolysis of double-caged IP₃ led to local calcium release. Uncaging of glycerophosphoryl-myo-inositol 4,5-bisphosphate (gPIP₂), a poorly metabolizable IP₃ analog, led to less well localized release. Thus, IP₃ breakdown is necessary for spine-specificity. IP₃- and gPIP₂-evoked signals declined from peak with similar, slow time courses, indicating that release lasts hundreds of milliseconds and is terminated not by IP₃ degradation but by intrinsic receptor dynamics. Based on measurements of spine-dendrite coupling, IP₃-evoked calcium signals are expected to be at least 2.4-fold larger in their spine of origin than in nearby spines, allowing IP₃ to act as a synapse-specific second messenger. Unexpectedly, single-caged IP₃ led to less release in somata and was ineffective in dendrites and spines. Calcium release using caged gPIP₂ was inhibited by the addition of single-caged IP₃, suggesting that single-caged IP₃ is an antagonist of calcium release. Caging at multiple sites may be an effective general approach to reducing residual receptor interaction.

Received for publication, October 13, 2006 , and in revised form, May 8, 2007.

^* This work was supported by a Whitehall Foundation grant, a W. M. Keck Foundation Young Investigator Award, National Science Foundation Grant 0347719, and National Institutes of Health Grant NS045193 (to S. S.-H. W.) and a Burroughs-Wellcome Interfaces of Science fellowship (to D. V. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Biology, Lewis Thomas Laboratory, Washington Rd., Princeton, NJ 08544. Tel.: 609-258-0388; Fax: 609-258-1028; E-mail: sswang{at}princeton.edu.

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