Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):E3301-10. doi: 10.1073/pnas.1217428110. Epub 2013 Aug 12.
cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca(2+) biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O2 can evoke a tonic increase in cGMP that requires an atypical O2-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca(2+) response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca(2+) stimulate competing negative feedback loops that shape cGMP dynamics. Ca(2+)-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O2, apparently by keeping in check gating of cGMP channels and limiting activation of Ca(2+)-dependent negative feedback loops. Simultaneous imaging of Ca(2+) and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O2-evoked cGMP and Ca(2+) responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.
环鸟苷酸信号广泛存在于神经系统中。然而，要在活体神经元中可视化和遗传探测内源性诱发的环鸟苷酸动态一直很困难。在这里，我们将环鸟苷酸和钙（Ca2+）生物传感器结合起来，在秀丽隐杆线虫的一个氧感应神经元中成像和剖析环鸟苷酸信号网络。我们表明，氧气的增加可以引发环鸟苷酸的持续增加，这需要一种非典型的氧结合可溶性鸟苷酸环化酶，并且这种增加会持续到氧气水平下降。增加的环鸟苷酸导致神经元中持续的 Ca2+反应，这依赖于环鸟苷酸门控离子通道。升高的环鸟苷酸和 Ca2+水平刺激竞争的负反馈回路，塑造环鸟苷酸动态。Ca2+依赖性负反馈回路，包括磷酸二酯酶-1（PDE-1）的激活，抑制环鸟苷酸的上升。一个不同的负反馈回路，由磷酸二酯酶-2（PDE-2）介导，并由环鸟苷酸依赖性激酶（PKG）刺激，出乎意料地促进了 O2 上升后环鸟苷酸的积累，显然是通过控制环鸟苷酸通道的门控和限制 Ca2+依赖性负反馈回路的激活来实现的。同时对 Ca2+和环鸟苷酸的成像表明，当同一动物的同一个神经元被重复刺激时，环鸟苷酸水平可以在接近环鸟苷酸通道的地方上升，而在其他地方下降。尽管动物在遗传上完全相同且受到类似的培养，但 O2 诱发的环鸟苷酸和 Ca2+反应在个体之间高度可重复，这表明环鸟苷酸转导具有很高的固有可靠性。然而，尽管动物在遗传上完全相同且受到类似的培养，但反应在个体之间差异很大。这种可变性可能反映了环鸟苷酸信号成分表达的随机差异。我们的工作为神经元中环鸟苷酸信号的结构提供了体内见解。
