Komal Pragya, Estakhr Jasem, Kamran Melad, Renda Anthony, Nashmi Raad
Department of Biology, Centre for Biomedical Research, University of Victoria, British Columbia, Canada.
J Physiol. 2015 Aug 15;593(16):3513-32. doi: 10.1113/JP270469. Epub 2015 Jun 25.
Protein kinases can modify the function of many proteins including ion channels. However, the role of protein kinase A in modifying nicotinic receptors in the CNS has never been investigated. We showed through whole-cell recordings of layer 1 prefrontal cortical interneurons that α7 nicotinic responses are negatively modulated by protein kinase A. Furthermore, we show that stimulation of dopamine receptors can similarly attenuate α7 nicotinic responses through the activation of protein kinase A. These results suggest how the interaction of the cholinergic and dopaminergic systems may influence neuronal excitability in the brain.
Phosphorylation of ion channels, including nicotinic acetylcholine receptors (nAChRs), by protein kinases plays a key role in the modification of synaptic transmission and neuronal excitability. α7 nAChRs are the second most prevalent nAChR subtype in the CNS following α4β2. Serine 365 in the M3-M4 cytoplasmic loop of the α7 nAChR is a phosphorylation site for protein kinase A (PKA). D1/D5 dopamine receptors signal through the adenylate cyclase-PKA pathway and play a key role in working memory and attention in the prefrontal cortex. Thus, we examined whether the dopaminergic system, mediated through PKA, functionally interacts with the α7-dependent cholinergic neurotransmission. In layer 1 interneurons of mouse prefrontal cortex, α7 nicotinic currents were decreased upon stimulation with 8-Br-cAMP, a PKA activator. In HEK 293T cells, dominant negative PKA abolished 8-Br-cAMP's effect of diminishing α7 nicotinic currents, while a constitutively active PKA catalytic subunit decreased α7 currents. In brain slices, the PKA inhibitor KT-5720 nullified 8-Br-cAMP's effect of attenuating α7 nicotinic responses, while applying a PKA catalytic subunit in the pipette solution decreased α7 currents. 8-Br-cAMP stimulation reduced surface expression of α7 nAChRs, but there was no change in single-channel conductance. The D1/D5 dopamine receptor agonist SKF 83822 similarly attenuated α7 nicotinic currents from layer 1 interneurons and this attenuation of nicotinic current was prevented by KT-5720. These results demonstrate that dopamine receptor-mediated activation of PKA negatively modulates nicotinic neurotransmission in prefrontal cortical interneurons, which may be a contributing mechanism of dopamine modulation of cognitive behaviours such as attention or working memory.
蛋白激酶可修饰包括离子通道在内的多种蛋白质的功能。然而，蛋白激酶A在中枢神经系统中对烟碱型受体的修饰作用从未被研究过。我们通过对前额叶皮层第1层中间神经元进行全细胞记录发现，α7烟碱型反应受到蛋白激酶A的负性调节。此外，我们还表明，多巴胺受体的刺激可通过激活蛋白激酶A同样减弱α7烟碱型反应。这些结果提示了胆碱能和多巴胺能系统的相互作用可能如何影响大脑中的神经元兴奋性。
蛋白激酶对离子通道（包括烟碱型乙酰胆碱受体，nAChRs）的磷酸化在突触传递和神经元兴奋性的修饰中起关键作用。α7 nAChRs是中枢神经系统中仅次于α4β2的第二普遍的nAChR亚型。α7 nAChR的M3-M4胞质环中的丝氨酸365是蛋白激酶A（PKA）的磷酸化位点。D1/D5多巴胺受体通过腺苷酸环化酶-PKA途径发出信号，在前额叶皮层的工作记忆和注意力中起关键作用。因此，我们研究了通过PKA介导的多巴胺能系统是否与α7依赖性胆碱能神经传递发生功能性相互作用。在小鼠前额叶皮层第1层中间神经元中，PKA激活剂8-溴-cAMP刺激后，α7烟碱型电流降低。在HEK 293T细胞中，显性负性PKA消除了8-溴-cAMP减弱α7烟碱型电流的作用，而组成型活性PKA催化亚基则降低了α7电流。在脑片中，PKA抑制剂KT-5720消除了8-溴-cAMP减弱α7烟碱型反应的作用，而在移液管溶液中应用PKA催化亚基则降低了α7电流。8-溴-cAMP刺激降低了α7 nAChRs的表面表达，但单通道电导没有变化。D1/D5多巴胺受体激动剂SKF 83822同样减弱了第1层中间神经元的α7烟碱型电流，并且KT-5720可阻止这种烟碱型电流的减弱。这些结果表明，多巴胺受体介导的PKA激活负性调节前额叶皮层中间神经元的烟碱型神经传递，这可能是多巴胺调节注意力或工作记忆等认知行为的一个促成机制。
