Gervasi Nicolas, Hepp Régine, Tricoire Ludovic, Zhang Jin, Lambolez Bertrand, Paupardin-Tritsch Danièle, Vincent Pierre
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7102, F-75005 Paris, France.
J Neurosci. 2007 Mar 14;27(11):2744-50. doi: 10.1523/JNEUROSCI.5352-06.2007.
The cAMP-dependent protein kinase A (PKA) plays a ubiquitous role in the regulation of neuronal activity, but the dynamics of its activation have been difficult to investigate. We used the genetically encoded fluorescent probe AKAR2 to record PKA activation in the cytosol and the nucleus of neurons in mouse brain slice preparations, whereas the potassium current underlying the slow afterhyperpolarization potential (sAHP) in thalamic intralaminar neurons was used to monitor PKA activation at the membrane. Adenylyl cyclase was stimulated either directly using forskolin or via activation of 5-HT7 receptors. Both stimulations produced a maximal effect on sAHP, whereas in the cytosol, the amplitude of the 5-HT7 receptor-mediated response was half of that after direct adenylyl cyclase stimulation with forskolin. 5-HT7-mediated PKA responses were obtained in 30 s at the membrane, in 2.5 min in the cytosol, and in 13 min in the nucleus. Our results show in morphologically intact mammalian neurons the potential physiological relevance of PKA signal integration at the subcellular level: neuromodulators produce fast and powerful effects on membrane excitability, consistent with a highly efficient functional coupling between adenylyl cyclases, PKA, and target channels. Phosphorylation in the cytosol is slower and of graded amplitude, showing a differential integration of the PKA signal between the membrane and the cytosol. The nucleus integrates these cytosolic signals over periods of tens of minutes, consistent with passive diffusion of the free catalytic subunit of PKA into the nucleus, eventually resulting in a graded modulation of gene expression.
环磷酸腺苷（cAMP）依赖性蛋白激酶A（PKA）在调节神经元活动中发挥着普遍作用，但其激活动力学一直难以研究。我们使用基因编码的荧光探针AKAR2记录小鼠脑片制备中神经元胞质溶胶和细胞核内的PKA激活情况，而丘脑层内神经元中慢后超极化电位（sAHP）的钾电流则用于监测膜上的PKA激活情况。通过直接使用福斯高林或激活5-HT7受体来刺激腺苷酸环化酶。两种刺激对sAHP均产生最大效应，而在胞质溶胶中，5-HT7受体介导的反应幅度是用福斯高林直接刺激腺苷酸环化酶后反应幅度的一半。5-HT7介导的PKA反应在膜上30秒内获得，在胞质溶胶中2.5分钟内获得，在细胞核中13分钟内获得。我们的结果表明，在形态完整的哺乳动物神经元中，PKA信号在亚细胞水平整合具有潜在的生理相关性：神经调质对膜兴奋性产生快速而强大的影响，这与腺苷酸环化酶、PKA和靶通道之间高效的功能偶联一致。胞质溶胶中的磷酸化较慢且幅度呈梯度变化，表明膜和胞质溶胶之间PKA信号的整合存在差异。细胞核在数十分钟内整合这些胞质溶胶信号，这与PKA的游离催化亚基被动扩散到细胞核中一致，最终导致基因表达的梯度调节。
