Novel isoforms of the sodium channels Nav1.8 and Nav1.5 are produced by a conserved mechanism in mouse and rat

Abstract

The voltage-gated sodium channel Na(v)1.8 is only expressed in subsets of neurons in dorsal root ganglia (DRG) and trigeminal and nodose ganglia. We have isolated mouse partial length Na(v)1.8 cDNA clones spanning the exon 17 sequence, which have 17 nucleotide substitutions and 12 predicted amino acid differences from the published sequence. The absence of a mutually exclusive alternative exon 17 was confirmed by sequencing 4.1 kilobases of genomic DNA spanning exons 16-18 of Scn10a. A novel cDNA isoform was identified, designated Na(v)1.8c, which results from alternative 3'-splice site selection at a CAG/CAG motif to exclude the codon for glutamine 1031 within the interdomain cytoplasmic loop IDII/III. The ratio of Na(v)1.8c (CAG-skipped) to Na(v)1.8 (CAG-inclusive) mRNA in mouse is approximately 2:1 in adult DRG, trigeminal ganglion, and neonatal DRG. A Na(v)1.8c isoform also occurs in rat DRG, but is less common. Of the two other tetrodotoxin-resistant channels, no analogous alternative splicing of mouse Na(v)1.9 was detected, whereas rare alternative splicing of Na(v)1.5 at a CAG/CAG motif resulted in the introduction of a CAG trinucleotide. This isoform, designated Na(v)1.5c, is conserved in rat and encodes an additional glutamine residue that disrupts a putative CK2 phosphorylation site. In summary, novel isoforms of Na(v)1.8 and Na(v)1.5 are each generated by alternative splicing at CAG/CAG motifs, which result in the absence or presence of predicted glutamine residues within the interdomain cytoplasmic loop IDII/III. Mutations of sodium channels within this cytoplasmic loop have previously been demonstrated to alter electrophysiological properties and cause cardiac arrhythmias and epilepsy.

Fig. 1. Amplification of Na_v1.8 cDNA from mouse DRG by RT-PCR

Products of the expected size were amplified from reverse-transcribed RNA of adult-control, adult-axotomy, and neonatal DRG (respectively, lanes 3, 5, and 7) equivalent to 20 ng of total RNA, but not from reverse-transcribed equivalents to 20 or 100 ng of total RNA from neonatal heart (respectively, lanes 9 and 11). No product was detected in corresponding RT-minus controls (respectively, lanes 4, 6, 8, 10, and 12). Lane 1 is a 1-kb DNA ladder (Invitrogen) showing fragments of 210–1018 bp, and lane 2 is blank.

Fig. 2. Alignment of the mouse, rat, and human Na_v1.8 predicted amino acid sequences within the IDII/III cytoplasmic loop

Predicted amino acid (aa) sequences are derived from mouse Na_v1.8 isoform cDNA (this report); the distal portion of exon 16 (88/151 aa) and the complete exon 17 (47 aa) of the mouse Scn10a gene (P70276, aa 943–1077; Ref. 36); rat cDNA (Na_v1.8/SNS, Q63554, aa 944–1078, Ref. ; Na_v1.8/PN3, Q62968, aa 943–1077, Ref. 4); and human cDNA (Na_v1.8/hPN3, Q9Y5Y9, aa 944–1076, Ref. 43). Arrows (↓) indicate predicted residues derived from mouse cDNA that differ from those of the reported mouse gene sequence, and the asterisk (*) indicates the glutamine residue (Q) not present in rodent Na_v1.8c isoforms due to alternative splicing (this report). Consensus residues conserved in mouse, rat, and human are shown in boldface, and a previously described putative ankyrin-G binding site is underscored that is highly similar in sequence and relative location to that necessary for Na_v1.5/ankyrin-G interaction (11, 26).

Fig. 3. Mouse and rat cDNA sequences of Na_v1.8 isoforms

A, a portion of mouse Na_v1.8 isoform cDNA sequence chromatogram (mNa_v1.8) is shown above those of mouse Na_v1.8c, rat Na_v(rNa_v1.8) and rat Na_v1.8c. Note that the Na_v1.8 isoforms each include a CAG trinucleotide (underlined) not present in the Na_v1.8c isoform sequences. The arrow indicates the junction of Na_v1.8 sequences transcribed from mouse Scn10a exons 16 and 17 (36), and rat sequence nucleotides differing from those of the mouse are also underlined. B, the mouse Na_v1.8d isoform retains the nucleotides GTAG from the 5′-end of intron 17. This is shown by comparing the Scn10a genomic DNA sequence (AJ622906, nt 1902–1916), where intron 17 nucleotides are lowercase, and the cryptic splice donor site GT dinucleotide is underlined, to the Na_v1.8d isoform cDNA sequence, which has an insertion of the nucleotides GTAG not found in the Na_v1.8 isoform cDNA sequence. The arrow indicates the junction of Na_v1.8 sequences transcribed from exons 17 and 18 (36).

Fig. 4. Amplification of Na_v1.5 cDNA isoforms from five mouse tissues by RT-PCR

A, products of the expected sizes for the Na_v1.5 and smaller Na_v1.5a isoform cDNAs were amplified from reverse-transcribed RNA of neonatal DRG (lane 3), neonatal heart (lane 5), adult heart (lane 7), and adult brain (lane 9). No apparent Na_v1.5 product was detected in reverse-transcribed adult thigh muscle (lane 11) or in the corresponding RT-minus controls (lanes 4, 6, 8, 10, and 12, respectively). Each 35-cycle run included an RT reaction equivalent to 20 ng of total RNA. Lane 1 is a 1-kb DNA ladder, and lane 2 is a water control. B, using RT reactions equivalent to 100 ng of total RNA in a 40-cycle run, products of the expected sizes for Na_v1.5 and Na_v1.5a cDNAs are detected in reverse-transcribed adult thigh muscle (lane 7), adult brain (lane 5), and neonatal DRG (lane 3), but not in the corresponding RT-minus controls (lanes 8, 6, and 4, respectively). Lane 1 is a 1-kb DNA ladder, and lane 2 is a water control.

Fig. 5. Mouse and rat cDNA sequences of Na_v1.5 and Na_v1.5c isoforms

The mouse Na_v1.5 isoform cDNA sequence (mNa_v1.5) is shown above those of mouse Na_v1.5c, rat Na_v1.5 (rNa_v1.5), and rat Na_v1.5c. Note that the Na_v1.5c isoform sequences each have an additional CAG trinucleotide (underlined) by comparison to the Na_v1.5 isoforms. Also underlined are the mouse silent nucleotide substitution G3284A (see text), and the trinucleotide ACA that distinguishes the rat sequence. Arrows indicate the junction of sequences transcribed from exons 17 and 18, by comparison to the human gene (38), which for visual clarity are only shown on the Na_v1.5c isoform sequences.

Fig. 6. The comparison of Na_v1.8 and Na_v1.5 predicted protein sequences from within the IDII/III cytoplasmic loop region

These topologically similar regions are encoded, respectively, by the splicing of mouse Scn10a exons 16/17 (36) and human SCN5A exons 17/18 (38), the relative junctions of which are indicated by forward slashes. A, the encoding cDNA sequences are from mouse (Na_v1.8 and Na_v1.8c, this report), rat (Na_v1.8/SNS/PN3, Refs. and ; Na_v1.8c, this report), and human (Na_v1.8/hPN3, Ref. 43). Note the absence of a conserved glutamine (Q) residue in the Na_v1.8c isoforms. The underlined mouse residue is P1035Q (see Table I). B, the encoding cDNA sequences are from human (Na_v1.5/hH1, Refs. and 71), mouse (Na_v1.5/mH1, Ref. ; Na_v1.5c, this report) and rat (Na_v1.5/rat heart I/SkM2, Refs. and ; Na_v1.5c, this report). Note the introduction of an additional glutamine (Q) residue in the rodent Na_v1.5c isoforms. Underlined residues represent putative CK2 phosphorylation site motifs present in rodent Na_v1.5 isoforms, but not in Na_v1.5c isoforms. The mouse and rat predicted protein sequences in this portion of Na_v1.5 are identical. Consensus residues conserved between mouse, rat and human are shown in boldface.