Alternative Splicing and CaV-Associated Channelopathies

Abstract

Voltage-gated calcium channels (VGCCs) are multi-subunit ion channel proteins that control and regulate a wide array of physiological processes. Their dysfunction has been implicated in several neurological, cardiac, psychiatric, endocrine, oncogenic, and muscular disorders. The diverse and specialized cellular functions involving VGCC-mediated calcium signaling stem from two primary mechanisms: differential and cell-specific expression of pore-forming (α1) and auxiliary subunit genes, and extensive alternative splicing of their pre-mRNA. All the 10 α1-encoding genes undergo alternative splicing to generate a wide array of cell-specific CaV variants with distinct biophysical, pharmacological, and protein-protein interaction properties. This proteomic diversity and the associated cell-specific expression signature of CaV splice variants are tightly regulated by trans-acting splicing factors-RNA-binding proteins that control the inclusion or skipping of alternatively spliced exons during post-transcriptional pre-mRNA processing. The discovery that several channelopathies are caused by aberrant splicing due to genetic mutations in either cis-acting binding elements on the pre-mRNA or in core splicing machinery components highlights the crucial role of alternative splicing in VGCC-related pathologies. These insights have opened new therapeutic avenues, as targeting the alternative splicing of disease-associated specific exons has recently emerged as a novel, promising treatment for neurodevelopmental disorders and channelopathies associated with splicing dysfunction.

Keywords: alternative splicing; calcium channel; disease; ion channel; therapy.

FIGURE 1

Context‐dependent control of alternative splicing by splicing factors. Splicing factors (SF) can promote the inclusion or skipping of alternatively spliced exons depending on their binding to intronic and exonic cis‐acting elements. (A) Splicing factors promote exon inclusion by binding to intronic splicing enhancer (ISE) sites on flanking introns or to exonic splicing enhancer (ESE) sites within an alternative exon. (B) Splicing factors promote exon skipping by binding to intronic splicing silencer (ISS) sites on flanking introns or to exonic splicing silencer (ESS) sites within an alternative exon. (C) Splicing factors can also act synergistically to promote alternative splicing. For instance, Nova and Ptbp2 cooperate to control the alternative splicing of a particular set of genes (Saito et al. 2019). Nova, Rbfox, and Mbnl proteins also work synergistically in the regulation of specific exons (Conboy 2017). (D) Antagonistic mechanism leading to the negative regulation of the action of a splicing factor by another. Nova can antagonize Ptbp2 splicing action by competing for Ptbp2 intronic binding sites on introns flanking Nova‐regulated exons, hence preventing Ptbp2 binding on those transcripts (Saito et al. 2019). Created in https://BioRender.com.

FIGURE 2

MNBL1 sequestering as a pathogenic mechanism in myotonic dystrophy‐1 (DM1). MBNL1 loss of function is caused by the high‐affinity binding of the protein to the expanded (CUG)n repeats (where > 50 repeats are pathogenic) on the 3′‐UTR of the dystrophia myotonic protein kinase 1 (DMPK1) RNA transcripts. Expansions of a CUG repeats‐containing segment, even up to several thousand base pairs, are found in patients with a severe form of the disease (Brook et al. 1992). MBNL1 promotes the inclusion of exon 29 in CaV1.1. MBNL1 sequestration causes aberrant splicing of exon 29, leading to the expression of a high‐conducting CaV1.1_∆29 variant in skeletal muscle with pronounced excitation–contraction (E–C) coupling. Created in https://BioRender.com.

FIGURE 3

RBFOX1 loss of function as a pathogenic mechanism in cardiac disorders. (Left) The CaV1.2_∆E9*,E33+ splice variant, which is generated from RBFOX1‐mediated inclusion of CACNA1C exon 33 and skipping exon 9*, aids normal heart physiology. (Right) In cardiac disorders, RBFOX1 loss of function leads to aberrant alternative splicing resulting in the abnormal inclusion of exon 9* and the skipping of exon 33 and in the expression of the CaV1.2_E9,ΔE33 splice variant that displays altered channel function and is arrhythmic. Created in https://BioRender.com.