Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions.

ABSTRACT: 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). 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+ on 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 (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 Ca2+ microenvironment around Cx36 gap junctions. Furthermore, HeLa cells can serve as a model system to study glutamate receptor-driven potentiation of electrical synapses.

SUBMITTER: Moore KB 

PROVIDER: S-EPMC7168262 | biostudies-literature | 2020 Mar/Apr

REPOSITORIES: biostudies-literature