Expanded alternative splice isoform profiling of the mouse Cav3.1/alpha1G T-type calcium channel

Abstract

Background: Alternative splicing of low-voltage-activated T-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. Transcript scanning of the human CACNA1G gene has revealed the presence of 11 regions within the coding sequence subjected to alternative splicing, some of which enhance T-type current. In mouse models of absence epilepsy, elevated T-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. To test whether enhanced T-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse Cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models.

Results: Transcript scanning of the murine Cacna1g gene detected 12 regions encoding alternative splice isoforms of Cav3.1/alpha1G T-type calcium channels. Of the 12 splice sites, six displayed homology to the human CACNA1G splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. In addition, two brain region-specific alternative splice patterns were observed in the cerebellum. Comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic T-type currents and wildtype controls failed to reveal differences in Cacna1g splicing patterns.

Conclusion: The determination of six novel alternative splice sites within the coding region of the mouse Cacna1g gene greatly expands the potential biophysical diversity of voltage-gated T-type channels in the mouse central nervous system. Although alternative splicing of Cav3.1/alpha1G channels does not explain the enhancement of T-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of T-type channels through this mechanism may influence other developmental neurological phenotypes.

Figure 1

Transcript scanning RT-PCR assay to detect alternative splicing events within the mouse Cacna1g gene. Schematic representation of the mouse Cacna1g pre-mRNA sequence and the RT-PCR primers (arrows, see Table 1) used for transcript scanning. Dashed grey lines denote the alternative splicing events and alternative splice donor and acceptor sites. Intron and exon sequences are not drawn to scale.

Figure 2

Identification of the mouse Cacna1g alternative splicing events. Cacna1g pre-mRNA undergoes extensive alternative splicing as detected by RT-PCR of mouse brain RNA samples. The spliced Cacna1g mRNA variants are illustrated to the right of each RT-PCR product: (A) Δ5' E2; (B) +I6-7 and Δ5' E8; (C) Δ3' E8; (D) ΔE12; (E) ΔE14; (F) Δ3' E25; (G) ΔE26; (H) Δ5' E31; (I) Δ5' E34 and ΔE34; and (J) ΔE35. Amplification of Cacna1g mRNA regions not undergoing alternative splicing was omitted. Lanes show the 100 bp ladder (L) and brain RT-PCR products (B). Intron and exon sequences are not drawn to scale.

Figure 3

Alternative splicing architecture of the mouse Cacna1g gene. (A) Representation of the Ca_v3.1/α1G T-type calcium channel structure and the localization of the 12 alternatively spliced regions: a) Δ5' E2; b) +I6-7; c) Δ5' E8; d) Δ3' E8; e) ΔE12; f) ΔE14; g) Δ3' E25; h) ΔE26; i) Δ5' E31; j) Δ5' E34; k) ΔE34; and l) ΔE35. Regions in black correspond to the previously identified alternative splice variants [9,12] and segments in grey denote the newly-characterized splice variants detected in this study. (B) Predicted protein sequences arising from the a), b), c), d), e), and j) alternative splicing events compared to full length Ca_v3.1/α1G protein, dashes indicate in-frame amino acid exclusion and the asterisk signifies a nonsense codon truncation.

Figure 4

Differential alternative splicing patterns detected in the adult mouse cerebellum. RT-PCR reactions from dissected wildtype and stargazer brain region RNAs (lanes: 1, thalamus; 2, cortex; 3, cerebellum; 4, hippocampus; +, wildtype control; -, heterozygous mutant) reveal differential splicing patterns at exons 14 and 26 in the cerebellum. Amplification of exon 14 shows preference in expression for the alternatively spliced product, ΔE14, at 623 bp, over the typically spliced product at 692 bp. The amplification of exon 26 identifies a shift in splicing resulting in sole expression of the alternatively spliced product, ΔE26, at 238 bp, while the normally spliced product at 292 bp is absent. The DNA marker on the far right separates at 100 bp intervals. Dissected lethargic and tottering samples show similar splicing patterns (data not shown).

Figure 5

lethargic mutant mice display unaltered Cacna1g mRNA alternative splicing within the brain. Compared to wildtype littermates (A), lethargic mutants (B) exhibit similar patterns of alternative splicing of Cacna1g transcripts assayed by RT-PCR from whole brain samples. Lanes within the agarose gel that are labelled (1–10) represent portions of the Cacna1g gene that undergo alternative splicing, while lanes without labels demonstrate regions of the gene not regulated by alternative splicing. Lane assignments: C, α-tubulin amplification loading control; 1, Δ5' E2; 2, +I6-7 & Δ5' E8; 3, Δ3' E8; 4, ΔE12; 5, ΔE14; 6, Δ3' E25; 7, ΔE26; 8, Δ5' E31; 9, Δ5' E34/ΔE34; 10, ΔE35. Alternative splice variants are denoted by the arrows.

Figure 6

tottering mutant mice display unaltered Cacna1g mRNA alternative splicing within the brain. Compared to wildtype littermates (A), tottering mutants (B) exhibit similar patterns of alternative splicing of Cacna1g transcripts assayed by RT-PCR from whole brain samples. Lanes within the agarose gel that are labelled (1–10) represent portions of the Cacna1g gene that undergo alternative splicing, while lanes without labels demonstrate regions of the gene not regulated by alternative splicing. Lane assignments: C, α-tubulin amplification loading control; 1, Δ5' E2; 2, +I6-7 & Δ5' E8; 3, Δ3' E8; 4, ΔE12; 5, ΔE14; 6, Δ3' E25; 7, ΔE26; 8, Δ5' E31; 9, Δ5' E34/ΔE34; 10, ΔE35. Alternative splice variants are denoted by the arrows.