Novel mRNA isoforms of the sodium channels Na(v)1.2, Na(v)1.3 and Na(v)1.7 encode predicted two-domain, truncated proteins

Abstract

The expression of voltage-gated sodium channels is regulated at multiple levels, and in this study we addressed the potential for alternative splicing of the Na(v)1.2, Na(v)1.3, Na(v)1.6 and Na(v)1.7 mRNAs. We isolated novel mRNA isoforms of Na(v)1.2 and Na(v)1.3 from adult mouse and rat dorsal root ganglia (DRG), Na(v)1.3 and Na(v)1.7 from adult mouse brain, and Na(v)1.7 from neonatal rat brain. These alternatively spliced isoforms introduce an additional exon (Na(v)1.2 exon 17A and topologically equivalent Na(v)1.7 exon 16A) or exon pair (Na(v)1.3 exons 17A and 17B) that contain an in-frame stop codon and result in predicted two-domain, truncated proteins. The mouse and rat orthologous exon sequences are highly conserved (94-100% identities), as are the paralogous Na(v)1.2 and Na(v)1.3 exons (93% identity in mouse) to which the Na(v)1.7 exon has only 60% identity. Previously, Na(v)1.3 mRNA has been shown to be upregulated in rat DRG following peripheral nerve injury, unlike the downregulation of all other sodium channel transcripts. Here we show that the expression of Na(v)1.3 mRNA containing exons 17A and 17B is unchanged in mouse following peripheral nerve injury (axotomy), whereas total Na(v)1.3 mRNA expression is upregulated by 33% (P=0.003), suggesting differential regulation of the alternatively spliced transcripts. The alternatively spliced rodent exon sequences are highly conserved in both the human and chicken genomes, with 77-89% and 72-76% identities to mouse, respectively. The widespread conservation of these sequences strongly suggests an additional level of regulation in the expression of these channels, that is also tissue-specific.

Fig. 1

The expression of Na_v1.2, Na_v1.3, Na_v1.6 and Na_v1.7 mRNAs in adult mouse DRG and brain. (A) RT-PCR analysis of sodium channel expression in adult mouse DRG showing larger, minor isoforms of Na_v1.2 and Na_v1.3. Products of the expected sizes for Na_v1.2 (lane 2), Na_v1.3 (lane 4), Na_v1.6 (lane 6) and Na_v1.7 (lane 8) were amplified from reverse-transcribed RNA, whereas no products were detected in the corresponding RT-minus controls (respectively, lanes 1, 3, 5 and 7). (B) RT-PCR analysis of sodium channel expression in adult mouse brain, showing larger, minor isoforms of Na_v1.3 and Na_v1.7 (shorter exposure shown in C). Lane designations in (B) and (C) are as in (A). M is 1 kb DNA ladder (Invitrogen) showing fragments of 1018, 517/506, 396, 344, 298 and 220/201 bp. The expected RT-PCR product sizes were 541 bp (Na_v1.2), 534 bp (Na_v1.3), 515 bp (Na_v1.6) and 575 bp (Na_v1.7).

Fig. 2

The alternatively spliced exon sequences of mouse and rat Na_v1.2, Na_v1.3 and Na_v1.7 mRNAs, showing in-frame termination codons (underlined bold). (A) Alignment of Na_v1.2 (Scn2a) exon 17A sequences of mouse (m) and rat (r), with conserved nt (m/r) indicated below by asterisks. (B) Na_v1.3 (Scn3a) exon 17A sequences; (C) Na_v1.3 (Scn3a) exon 17B sequences; and (D) Na_v1.7 (Scn9a) exon 16A sequences. (E) Alignments of the mouse Na_v1.2, Na_v1.3 and Na_v1.7 alternatively spliced exon sequences, with conserved nt between Na_v1.2 and Na_v1.3 (m1.2/1.3) and between Na_v1.3 and Na_v1.7 (m1.3/1.7) shown, respectively, above and below by asterisks. The downward arrow in (B) and (E) indicates the site of the relative deletion of a single nucleotide in mouse Na_v1.3 exon 17A that results in the reading frame extending downstream into exon 17B, and ‘-’ indicates a gap introduced to optimize an alignment.

Fig. 3

Schematic diagram of the mouse and rat Scn2a (Na_v1.2), Scn3a (Na_v1.3) and Scn9a (Na_v1.7) genes in the regions of the novel alternatively spliced exons. Exons are represented by boxes and introns by lines (not to scale), with constitutive exons numbered as previously published (Kasai et al., 2001; Yang et al., 2004; Cox et al., 2006) and in-frame stop codons within the alternatively spliced exons of mouse and rat indicated above and below, respectively. Intron lengths of the mouse and rat genes are in kb, and canonical GT-AG dinucleotides present at the donor and acceptor splice sites in both mouse and rat are shown. The downstream donor splice site selections at exons 17A that can result in nine nucleotide extensions of the rat Na_v1.2 and mouse Na_v1.3 mRNAs are indicated by dotted lines. Shown below is the predicted topology of a generic voltage-gated sodium channel α-subunit within the plasma membrane (gray), with arrows indicating the corresponding predicted termination sites (X, stop codons) within the second cytoplasmic loop that result from alternative splicing of mouse and rat (m/r) Na_v1.2, Na_v1.3 and Na_v1.7 mRNAs. The alternatively spliced mouse Na_v1.3 mRNA isoforms encode either 34 or 37 novel C-terminal amino acids.

Fig. 4

The expression of Na_v1.3 mRNA isoforms in adult mouse DRG 1 week after axotomy. In quantitative RT-PCR assays the expression of Na_v1.3 mRNA containing exons 17A and 17B (17A+B) was unchanged (n=5; 0.810±0.049 of control, not significant with P>0.05), total Na_v1.3 mRNA increased to 1.328±0.030 of control (n=5; P=0.003) and Na_v1.6 mRNA decreased to 0.586±0.025 of control (n=5; P<0.001) in pooled ipsilateral (axotomized) lumbar L4 and L5 DRG compared with contralateral (unaxotomized) controls 1 week after axotomy. Data are shown as means±S.E., in which expression after axotomy (filled boxes) was compared with contralateral controls of 1.00 relative units (unfilled boxes). Two asterisks, P<0.005; three asterisks, P<0.001.

Fig. 5

The expression of Na_v1.2, Na_v1.3, Na_v1.6 and Na_v1.7 mRNAs in adult rat DRG and neonatal brain. (A) RT-PCR analysis of sodium channel expression in adult rat DRG showing larger, minor isoforms of Na_v1.2 and Na_v1.3 (shorter exposure shown in B). (C) RT-PCR analysis of sodium channel expression in neonatal rat brain showing a larger isoform of Na_v1.7 (lane 8), not detected in adult rat brain (lane 10). Lane designations and DNA ladder fragment sizes are as in Fig. 1, except for (C) adult (ad) brain lanes 9 (RT-minus, Na_v1.7) and 10 (RT-plus, Na_v1.7). An erratic band of ~300 bp detectable in some rat samples amplified with Na_v1.6 primers is a misprimed product unrelated to sodium channels (data not shown). The expected RT-PCR product sizes were 474 bp (Na_v1.2), 505 bp (Na_v1.3), 519 bp (Na_v1.6) and 537 bp (Na_v1.7).

Fig. 6

The human and chicken putative exon sequences with high levels of conservation to the alternatively spliced exons of mouse and rat Na_v1.2 (Scn2a), Na_v1.3 (Scn3a) and Na_v1.7 (Scn9a) mRNAs. The sequences of the human (h) and chicken (ch) putative exon 17A of SCN2A (A), exon 17A of SCN3A (B), exon 17B of SCN3A (C) and exon 16A of SCN9A (D) are each shown with conserved nt between human and mouse (h/m) and between chicken and mouse (ch/m) indicated, respectively, above and below by asterisks. Note that no sequence homologous to SCN3A exon 17B was detected in chicken. Chicken gene designations for SCN2A follow locus 395945 and published work (Striano et al., 2006; Martin et al., 2007); SCN3A follows Martin et al. (2007) and is currently listed as locus 424180 in genome build 2.1; and for SCN9A, see Discussion. Underlined bold capitals are putative in-frame termination codons, and ‘-’ indicates a gap introduced to optimize an alignment.