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Sheila A Baker, Vasily Kerov, Joseph G. Laird, Brittany Williams, Mei-Ling Joiner, Amy Lee; The role of Cav1.4 calcium channels in photoreceptor synapse development. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1325.
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© ARVO (1962-2015); The Authors (2016-present)
Cav1.4 is the voltage-gated calcium channel localized beneath the synaptic ribbon in rods and cones. It mediates the inward flux of Ca2+ current that triggers neurotransmitter release in the dark adapted photoreceptor. Loss of Cav1.4 function presents in a variety of disorders including congenital stationary night blindness, cone-rod dystrophy, retinitis pigmentosa and Leber’s congenital amaurosis. Altering the function of Cav1.4 cripples neurotransmission but it also prevents the development or maintenance of the photoreceptor synaptic ribbon. The goal of this study was to identify molecular mechanisms by which Cav1.4 regulates synaptogenesis.
In vivo electroporation was used to transfect rods of Cav1.4 knockout mice with FLAG-tagged wildtype (WT) Cav1.4 or mutants of the channel. Electroporations were conducted on neonates (P0) and analysis was conducted on young adults beginning after eye opening, P14 to P30. Transfected photoreceptors were identified based on co-expression of a fluorescent marker and immunostaining for FLAG. Synapses were considered rescued if the presynaptic membrane marker, PSD-95, was expressed, RIBEYE labeled ribbons were elongated, and the ribbons were opposed to mGluR6 labeled bipolar dendritic tips. Whole-cell patch clamp recording of FLAG-Cav1.4 transfected HEK cells was used to verify the biophysical properties of the mutant channels.
Restoring expression of FLAG-Cav1.4-WT rescued synaptic development in transfected Cav1.4 knockout rods. A non-conducting channel mutant with three pore-lining glutamates mutated to glutamines, FLAG-Cav1.4-3EQ, also rescued synaptic development. However, ribbons associated with FLAG-Cav1.4 3EQ were shorter than ribbons associated with FLAG-Cav1.4 WT. A channel with a disrupted voltage-sensor, FLAG-Cav1.4-F137A, rescued synaptic development.
Exogenous expression of Cav1.4 was sufficient to restore morphology of the synapse in Cav1.4 knockout rods. Since there are many disease-causing mutations in Cav1.4 that disrupt Ca2+ signaling, it was surprising that the mutants studied here could support synaptogenesis. These data lead to a model in which Cav1.4 has a scaffolding role in the initial development of the ribbon synapse whereas proper regulation of its activity is required for maintenance. This work provides insight into the pathogenesis of numerous synaptic diseases in the retina.
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