Richard S. Ruiz, M.D. Department of Ophthalmology and Visual Science, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030.
The MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030.
eNeuro. 2020 Apr 17;7(2). doi: 10.1523/ENEURO.0445-19.2020. Print 2020 Mar/Apr.
A variety of electrical synapses are capable of activity-dependent plasticity, including both activity-dependent potentiation and activity-dependent depression. In several types of neurons, activity-dependent electrical synapse plasticity depends on changes in the local Ca environment. To enable study of local Ca signaling that regulates plasticity, we developed a GCaMP Ca biosensor fused to the electrical synapse protein Connexin 36 (Cx36). Cx36-GCaMP transfected into mammalian cell cultures formed gap junctions at cell-cell boundaries and supported Neurobiotin tracer coupling that was regulated by protein kinase A signaling in the same way as Cx36. Cx36-GCaMP gap junctions robustly reported local Ca increases in response to addition of a Ca ionophore with increases in fluorescence that recovered during washout. Recovery was strongly dependent on Na-Ca exchange activity. In cells transfected with NMDA receptor subunits, Cx36-GCaMP revealed transient and concentration-dependent increases in local Ca on brief application of glutamate. In HeLa cells, glutamate application increased Cx36-GCaMP tracer coupling through a mechanism that depended in part on Ca, calmodulin-dependent protein kinase II (CaMKII) activity. This potentiation of coupling did not require exogenous expression of glutamate receptors, but could be accomplished by endogenously expressed glutamate receptors with pharmacological characteristics reminiscent of NMDA and kainate receptors. Analysis of RNA Sequencing data from HeLa cells confirmed expression of NMDA receptor subunits NR1, NR2C, and NR3B. In summary, Cx36-GCaMP is an effective tool to measure changes in the Ca microenvironment around Cx36 gap junctions. Furthermore, HeLa cells can serve as a model system to study glutamate receptor-driven potentiation of electrical synapses.
多种电突触具有活动依赖性可塑性，包括活动依赖性增强和活动依赖性抑制。在几种类型的神经元中，活动依赖性电突触可塑性取决于局部 Ca 环境的变化。为了能够研究调节可塑性的局部 Ca 信号，我们开发了一种与电突触蛋白 Connexin 36（Cx36）融合的 GCaMP Ca 生物传感器。Cx36-GCaMP 转染到哺乳动物细胞培养物中，在细胞-细胞边界处形成缝隙连接，并支持神经生物素示踪剂偶联，这种偶联受蛋白激酶 A 信号的调节方式与 Cx36 相同。Cx36-GCaMP 缝隙连接在加入 Ca 离子载体时会强烈报告局部 Ca 增加，荧光强度增加，在冲洗过程中恢复。恢复强烈依赖于 Na-Ca 交换活性。在转染 NMDA 受体亚基的细胞中，Cx36-GCaMP 揭示了短暂的、浓度依赖性的局部 Ca 增加，这是在短暂应用谷氨酸时发生的。在 HeLa 细胞中，谷氨酸应用通过部分依赖 Ca、钙调蛋白依赖性蛋白激酶 II（CaMKII）活性的机制增加 Cx36-GCaMP 示踪剂偶联。这种偶联的增强不需要外源性表达谷氨酸受体，但可以通过具有类似于 NMDA 和海人藻酸受体的药理学特征的内源性表达谷氨酸受体来完成。来自 HeLa 细胞的 RNA 测序数据的分析证实了 NMDA 受体亚基 NR1、NR2C 和 NR3B 的表达。总之，Cx36-GCaMP 是测量 Cx36 缝隙连接周围 Ca 微环境变化的有效工具。此外，HeLa 细胞可以作为研究谷氨酸受体驱动的电突触增强的模型系统。
