Sarkisov Dmitry V, Gelber Shari E, Walker Jeffery W, Wang Samuel S-H
Department of Physics and Molecular Biology and Program in Neuroscience, Princeton University, Princeton, New Jersey 08544, USA.
J Biol Chem. 2007 Aug 31;282(35):25517-26. doi: 10.1074/jbc.M609672200. Epub 2007 May 31.
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.
通过添加单个光敏基团可以“笼化”生物信使，该光敏基团可通过光脉冲进行光解，从而实现空间和时间上精确的生化控制。在此，我们报告，通过双光子显微镜测量发现，第二信使肌醇1,4,5-三磷酸（IP3）的双笼形式的光解触发了浦肯野细胞胞体、树突和棘突中的局灶性钙释放。在钙结合蛋白基因敲除的浦肯野细胞中，峰值钙随闪光能量增加，双笼化IP3比传统单笼化IP3具有更高的协同性，这与化学双光子效应一致。双笼化IP3的棘突光解导致局部钙释放。甘油磷酸肌醇4,5-二磷酸（gPIP2），一种代谢缓慢的IP3类似物的解笼化导致定位较差的释放。因此，IP3的分解对于棘突特异性是必要的。IP3和gPIP2诱发的信号从峰值开始以相似的缓慢时间进程下降，表明释放持续数百毫秒，并且不是由IP3降解而是由内在受体动力学终止。基于对棘突-树突耦合的测量，预计IP3诱发的钙信号在其起源的棘突中比在附近棘突中至少大2.4倍，这使得IP3能够作为突触特异性第二信使。出乎意料的是，单笼化IP3在胞体中导致较少的释放，并且在树突和棘突中无效。添加单笼化IP3可抑制使用笼化gPIP2的钙释放，表明单笼化IP3是钙释放的拮抗剂。在多个位点进行笼化可能是减少残余受体相互作用的一种有效的通用方法。
