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John O'Brien, Ya-Ping Lin, Wade Kothmann, Keith B Moore, Cheryl K Mitchell, Jeffrey S Diamond; Calcium signaling at Cx36 gap junctions in the mouse retina. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Electrical synapses made of Cx36 are capable of potentiation via a Ca2+-Calmodulin-CaM kinase II signaling pathway. This has been observed to date in goldfish Mauthner neurons, mammalian AII amacrine cells and inferior olive neurons. We hypothesize that such signaling is widespread.
To study Ca2+ signaling we developed a Cx36-GCaMP3 fusion protein and transgenic mice expressing Cx36-GCaMP driven by a 5 kb Cx36 promoter. We examined Ca2+ signaling by optical imaging and functionality by tracer coupling in transfected mammalian cells. We examined Ca2+ signals in response to glutamate puffs in retina slice preparations from Cx36-GCaMP transgenic mice by optical imaging.
Cx36-GCaMP transfected in mammalian cells formed junctional plaques and generated strong, transient fluorescence increases upon addition of ionomycin. Cx36-GCaMP supported tracer coupling in HeLa cells that was regulated by PKA activity in the same manner as wild type Cx36. In HEK cells co-transfected with NMDA receptor subunits NR1 plus either NR2A, NR2B or NR2C, Cx36-GCaMP produced transient increases in fluorescence upon perfusion of glutamate. In slices of retina from Cx36-GCaMP transgenic mice, approximately half of fluorescent gap junctions imaged in the inner plexiform layer responded to 2 sec puffs of glutamate with fluorescence increases lasting 10-15 sec. Responses varied greatly in magnitude with many individual gap junctions responding with peak F/Fo > 3. Co-application of the NMDA receptor antagonist CPP blocked or strongly attenuated some of those responses, significantly reducing the average peak response.
Many gap junctions in the retina experience transient Ca2+ increases dependent on NMDA receptors. Some also experience Ca2+ increases of a different origin. This Ca2+ may regulate coupling via CaMKII signaling.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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