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Abstract：
Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle. The resulting temporal control over melanopsin activation allowed us to compare the activation kinetics of different components of the electrophysiological response. We also replaced the intracellular loops of melanopsin with those of the 5-HT2A receptor to create a light-activated GPCR capable of interacting with the 5-HT2A receptor interacting proteins. The resulting chimera expressed weak activity but validated the potential usefulness of melanopsin as a tool for the study of G protein signaling in CNS neurons.
摘要：
我们对中枢神经系统（CNS）中G蛋白偶联受体（GPCRs）的理解受到工具有限的限制，这些工具可以通过精确的时间控制来研究其信号传导。为了克服这一点，我们测试了双稳态哺乳动物视蛋白黑视蛋白检查CNS神经元中G蛋白信号的实用性。具体来说,我们使用生物射弹（基因枪）方法将黑视素转染到器官切片培养中维持的皮质锥体细胞中。来自转染神经元的全细胞记录表明，蓝光的应用有效地激活了转染的黑视素，以引起先前与GPCRs偶联到Gαq-11的激活相关的膜兴奋性的经典双相调节。可以用短至几毫秒的脉冲获得外源激动剂浓度的完全模拟，这表明它们的触发需要一个单一的黑视素激活-失活循环。对黑视素激活的时间控制使我们能够比较电生理反应不同成分的激活动力学。我们还用5-HT2A受体的那些取代了黑视素的胞内环,以产生能够与5-HT2A受体相互作用蛋白相互作用的光活化GPCR。所得的嵌合体表达弱活性，但验证了黑视素作为研究CNS神经元中G蛋白信号传导的工具的潜在有用性。
