TY - JOUR T1 - Localized calcium signaling and the control of coupling at Cx36 gap junctions JF - eneuro JO - eNeuro DO - 10.1523/ENEURO.0445-19.2020 SP - ENEURO.0445-19.2020 AU - Keith B. Moore AU - Cheryl K. Mitchell AU - Ya-Ping Lin AU - Yuan-Hao Lee AU - Eyad Shihabeddin AU - John O’Brien Y1 - 2020/03/11 UR - http://www.eneuro.org/content/early/2020/03/10/ENEURO.0445-19.2020.abstract N2 - 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 Ca2+ environment. To enable study of local Ca2+ signaling that regulates plasticity, we developed a GCaMP Ca2+ biosensor fused to the electrical synapse protein Connexin 36. 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 Ca2+ increases in response to addition of a Ca2+ ionophore with increases in fluorescence that recovered during washout. Recovery was strongly dependent on Na+-Ca2+ exchange activity. In cells transfected with NMDA receptor subunits, Cx36-GCaMP revealed transient and concentration-dependent increases in local Ca2+ upon brief application of glutamate. In HeLa cells, glutamate application increased Cx36-GCaMP tracer coupling through a mechanism that depended in part on Ca2+, calmodulin-dependent protein kinase II 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 Ca2+ microenvironment around Cx36 gap junctions. Furthermore, HeLa cells can serve as a model system to study glutamate receptor-driven potentiation of electrical synapses.Significance We have developed a Connexin 36-GCaMP3 fusion construct that effectively reports the Ca2+ microenvironment in the vicinity of Cx36 gap junctions. This tool will be valuable to investigate the dynamic changes in Ca2+ that are responsible for some forms of electrical synapse plasticity. Furthermore, we have discovered that a widely used model system for in vitro studies, the HeLa cell, endogenously expresses glutamate receptors that effectively drive intracellular Ca2+, calmodulin-dependent protein kinase II signaling. This signaling can be exploited in many types of studies. ER -