A New Splicing Isoform of Cacna2d4 Mimicking the Effects of c.2451insC Mutation in the Retina: Novel Molecular and Electrophysiological Insights

作者：Bacchi Niccolo; Messina Andrea; Burtscher Verena; Dassi Erik; Provenzano Giovanni; Bozzi Yuri; Demontis Gian Carlo; Koschak Alexandra; Denti Michela A; Casarosa Simona^*

来源：Investigative Ophthalmology & Visual Science, 2015, 56(8): 4846-4856.

DOI：10.1167/iovs.15-16410

• Ca2+ channels
• exon-skipping
• splicing isoforms
• Cacna2d4

PURPOSE. Mutations in CACNA2D4 exon 25 cause photoreceptor dysfunction in humans (c. 2406C -> A mutation) and mice (c. 2451insC mutation). We investigated the feasibility of an exon-skipping therapeutic approach by evaluating the splicing patterns and functional role of targeted exons.
METHODS. Splicing of the targeted alpha(2)delta(4) (CACNA2D4) exons in presence and absence of the mutation was assessed by RT-PCR in vivo on mouse retinae and in vitro in HEK293T cells using splicing-reporter minigenes. Whole-cell patch-clamp recordings were performed to evaluate the impact of different Cacna2d4 variants on the biophysical properties of Cav1.4 L-type calcium channels (CACNA1F).
RESULTS. Splicing analysis revealed the presence of a previously unknown splicing isoform of alpha(2)delta(4) in the retina that truncates the gene open reading frame (ORF) in a similar way as the c. 2451insC mutation. This isoform originates from alternative splicing of exon 25 (E25) with a new exon (E25b). Moreover, the c. 2451insC mutation has an effect on splicing and increases the proportion of transcripts including E25b. Our electrophysiological analyses showed that only full-length alpha(2)delta(4) was able to increase Cav1.4/beta 3-mediated currents while all other alpha(2)delta(4) variants did not mediate such effect.
CONCLUSIONS. The designed exon-skipping strategy is not applicable because the resulting skipped alpha(2)delta(4) are nonfunctional. alpha(2)delta(4) E25b splicing variant is normally present in mouse retina and mimics the effect of c. 2451insC mutation. Since this variant does not promote significant Cav1.4-mediated calcium current, it could possibly mediate a different function, unrelated to modulation of calcium channel properties at the photoreceptor terminals.

• 出版日期2015-7