title: A New Splicing Isoform of Cacna2d4 Mimicking the Effects of c.2451insC Mutation in the Retina: Novel Molecular and Electrophysiological Insights.
abstract: OBJECTIVE
  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 α2δ4 (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 α2δ4 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 α2δ4 was able to increase Cav1.4/β3-mediated currents while all other α2δ4 variants did not mediate such effect.
  
  
  CONCLUSIONS
  The designed exon-skipping strategy is not applicable because the resulting skipped α2δ4 are nonfunctional. α2δ4 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.
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Niccolò
authors.initials: N
authors.lastname: Bacchi
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Messina
authors.affiliation: Medical University of Vienna, Center for Physiology and Pharmacology, Department of Neurophysiology and Pharmacology, Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Burtscher
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Erik
authors.initials: E
authors.lastname: Dassi
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Provenzano
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy 3Neuroscience Institute-National Research Council (CNR), Pisa, Italy.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Bozzi
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: Gian Carlo
authors.initials: GC
authors.lastname: Demontis
authors.affiliation: Medical University of Vienna, Center for Physiology and Pharmacology, Department of Neurophysiology and Pharmacology, Vienna, Austria 5University of Innsbruck, Institute of Pharmacy, Pharmacology and Toxicology, Center for Chemistry and Biomedicine, Innsb.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Koschak
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy 6Neuroscience Institute-National Research Council (CNR), Padova, Italy.
authors.forename: Michela A
authors.initials: MA
authors.lastname: Denti
authors.affiliation: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy 3Neuroscience Institute-National Research Council (CNR), Pisa, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Casarosa
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D020746:Calcium Channels, L-Type / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D005091:Exons; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009154:Mutation; D018408:Patch-Clamp Techniques; D012313:RNA / Q000235:genetics*; D012326:RNA Splicing; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D058499:Retinal Dystrophies / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C512890:Cacna2d4 protein, mouse; D020746:Calcium Channels, L-Type; D012313:RNA