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Domino Schlegel, Stella Glasauer, Stephan C F Neuhauss; Functional Characterization of the Calcium Channel Subunit Cacna2d4 in the Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4486.
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© ARVO (1962-2015); The Authors (2016-present)
Voltage-gated calcium channels are heteromultimeric complexes consisting of a pore-forming α1 subunit and auxiliary β and α2δ subunits. These complexes are expressed in excitable cells and open upon membrane depolarization, thereby leading to an influx of Ca2+ which in turn triggers different Ca2+-dependent processes. In photoreceptor cells, the calcium channel subtype 1.4 is crucial for signal transmission to second order neurons by mediating glutamate release into the synaptic cleft. While the α1 subunit is essential for this process, the auxiliary α2δ subunit is thought to have a more indirect role in neurotransmitter release by targeting the pore-forming subunit to the cell membrane as well as by modulating the channel’s biophysical properties. Although the function of the retina-specific subtype α2δ-4 has not yet been described in detail, patients carrying mutations in the respective gene, CACNA2D4, exhibit symptoms of cone-rod dysfunction. Therefore we aim to investigate the mechanisms underlying the onset and progression of the disease by using zebrafish as a model system.
We made use of the cone-dominant zebrafish to study the molecular mechanism of the dysfunction in CRISPR/Cas9-knockout lines of the respective paralogs cacna2d4a and cacna2d4b by histological and electrophysiological analysis.
We show that absence of Cacna2d4b, but not Cacna2d4a, leads to reduced expression as well as mislocalization of Cacna1fa, the pore-forming subunit in zebrafish, suggesting an involvement of Cacna2d4b in targeting the channel to the synaptic terminal. However, we could not detect changes in the b-wave amplitude of the electroretinogram (ERG) in cacna2d4b-/- larvae at 5 days post fertilization. Only in double-knockout (cacna2d4a-/-;b-/-) fish did we find a reduction in the b-wave amplitude in addition to impaired expression of the α1 subunit, pointing towards compensatory mechanisms involving cacna2d4a in cacna2d4b-/- single knockout. Interestingly, in cacna2d4a-/-;b-/- retinae Ribeyeb localization is affected and abundance of the postsynaptic glutamate transporter EAAT7 is reduced.
Our findings suggest that Cacna2d4b is involved in trafficking of the pore-forming subunit. Furthermore, we show that lack of both paralogues reduces expression of Cacna1fa at the synapse, and causes an impaired ERG response as well as pre- and postsynaptic alterations.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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