RNA isolation, cDNA library construction, and screening

Total RNA was extracted from the retina of adult newts (Cynops pyrrhogaster) using the guanidinium isothiocyanate method (RNA Extraction Kit, Pharmacia Biotech, Piscataway, NJ). Poly(A)⁺ RNA was isolated by performing oligo (dT)-cellulose chromatography twice (mRNA Purification Kit, Pharmacia Biotech). The cDNAs were synthesized from either oligo(dT) or a random primer, ligated into λZAP (Stratagene, La Jolla, CA), and transfected into XL-1 Blue cells. The transfected cells were plated over plastic culture dishes to form plaques and transferred onto nylon membrane filters (Hybond-N; Amersham, Arlington Heights, IL). Hybridization was performed at 65°C for 16 hr in 6×SSC, 5×Denhardt's solution, 0.5% SDS, 20 μg/ml Salmon sperm DNA, and ³²P-labeled probe. After hybridization, the filters were washed in 0.2×SSC at 65°C for 20 min, and analyzed using a bio-image analyzer (BAS2000; Fuji Photo Film, Japan). Plaque-purified clones were rescued as pBluescript phagemids and sequenced using an ABI 377 automated DNA sequencer (Perkin-Elmer, Foster City, CA).

The oligo(dT)-primed library was screened with a cDNA probe encoding a highly conserved region of the repeat IV (P_IV), and the random-primed library was screened with a probe of the repeat I (P_I). The two probes were obtained by RT-PCR using the following primer pairs designed from the nucleotide sequence of the rat brain Na⁺ channel; P_I, 5′-CC[A/T]CTGGAGGACCTGGACCCCTACTAT-3′ (IS) corresponding to the peptide ₇₆PLEDLDPYY and 5′-TGTGGCCTGGTTCTGTTCCTC[A/G]TAGGC-3′ (IAS) corresponding to the peptide ₄₂₅AYEEQNQAT; P_IV, 5′-CTGGTGTTCATTGTGCTGTTCACCGGC-3′ (IVS) corresponding to the peptide ₁₅₇₈LVFIVLFTG and 5′-GACAGCGATGTACATGTTCACCACCAC-3′ (IVAS) corresponding to the peptide ₁₇₇₆VVVNMYIAV of the rat brain type I Na⁺ channel (Noda et al., 1986). After the reverse transcriptase step was carried out at 42°C for 15 min, a PCR reaction containing 200 nM of each primer was performed using a GeneAmp RNA PCR Kit (Perkin-Elmer, Norwalk, CT) under the conditions recommended by the manufacturer. The PCR amplification consisted of 105 sec at 95°C, 35 cycles of 15 sec at 95°C, 30 sec at 60°C and a final 7 min at 72°C. The PCR products were cloned into pCR^TMII vector using a TA-cloning Kit (Invitrogen, San Diego, CA), and sequenced.

RT-PCR amplification and cloning

cDNA spanning the blank region between the clone from the random-primed library and the clone from the oligo(dT)-primed library was generated by RT-PCR using PfuTurbo DNA polymerase (Stratagene), which has 3′–5′ exonuclease activity (proofreading activity). First strand cDNA was synthesized from poly(A) RNA with oligo(dT) primer at 42°C for 15 min using a GeneAmp RNA PCR Kit (Perkin Elmer). The PCR reaction was performed in a 100 μl reaction solution with 200 nM each primer under the conditions recommended by the manufacturer (Stratagene). The amplification consisted of 1 min at 95°C, 30 cycles of 1 min at 95°C, 1 min at 60°C, 8 min 72°C, and a final 10 min at 72°C. The second PCR reaction was performed using 1 μl of the first PCR product for 20 cycles under the same thermal conditions. The sense primer (PS) was 5′-GGCACAGAAACAGAAATGAAGAAG-3′, corresponding to nt 2,244–2,267, and the antisense primer (PAS) was 5′-ATGCCATAAATCGTTCTTCCATTG-3′, complementary to nt 5866-5889. Agarose gel electrophoresis of the PCR product showed a single band of the expected size. Then, the PCR product was excised from the agarose gel, purified, cloned into pPCR-Script vector using a PCR-Script Amp Cloning Kit (Stratagene), and sequenced.

In situ hybridization

The cDNA fragment corresponding to nt 118–1,197 was obtained by RT-PCR, cloned into pCR^TMII vector using a TA-cloning Kit (Invitrogen), and confirmed by sequencing. The plasmids were linearized and transcribed with T7 or SP6 RNA polymerase separately, to obtain both antisense and sense probes. The probes were labeled with digoxigenin-labeled nucleotides using a DIG RNA Labeling Kit (Boehringer Mannheim, Indianapolis IN). To prepare newt retina cryosections, after removing the cornea and lens, the eye cups were fixed with 4% paraformaldehyde in PBS overnight at 4°C, cryoprotected by equilibration with 30% sucrose in PBS, and embedded in OCT compound (Tissue-Tek; Miles Inc., Elkhart, IN). 15 μm sections were cut on a cryostat at −20°C and mounted on gelatin-coated slides. The sections were treated with 10 μg/ml proteinase K (Boehringer Mannheim) for 10 min at 37°C, and then fixed with 4% paraformaldehyde in PBS for 30 min. Hybridization was carried out by incubation with probe (0.7 μg/ml) overnight at 50°C. Then, the sections were washed several times and treated with 20 μg/ml RNase A (QIAGEN Inc., Chatsworth, CA) in 500 mM NaCl, 10 mM Tris-HCl (pH8.0) for 30 min at 37°C, with final washes in 1×SSC, 50% formamide for 30 min at 45°C and for 30 min at room temperature. Digoxigenin was detected with alkaline phosphatase-labelled anti-digoxigenin antibodies using a Digoxigenin Detection Kit (Boehringer Mannheim). The specimens were examined and photographed using a transmitted light microscope (DMR; Leica Instruments, Germany).

Isolation and primary structure of the newt retina Na⁺ channel

The cDNAs encoding highly conserved regions of repeats I and IV were amplified by RT-PCR with specific primer sets, [IS, IAS] and [IVS, IVAS], respectively. PCR products of the expected sizes were seen as single bands in 1% agarose gels. These were excised from the gels, and cloned into vector. Sequencing the DNA revealed a high sequence similarity to known Na⁺ channels. Specifically, P_I, the product from primer pair [IS, IAS], had a deduced amino acid sequence similar (83%) to that in the repeat I of the rat brain type I Na⁺ channel, and P_IV, the product from primer pair (IVS, IVAS), was similar (93%) to the repeat IV. These were used as probes for library screening.

One cDNA clone (R1), which had an identical sequence to the probe (P_I) in their overlapping region, was isolated from the random-primed cDNA library using P_I. One clone (T1) isolated from the oligo(dT)-primed cDNA library using P_IV also had an identical nucleotide sequence to the probe in their overlapping region. The rest of the coding region of the Na⁺ channel between R1 and T1 was obtained by RT-PCR using PfuTurbo DNA polymerase (Stratagene), which has high 3′–5′ exonuclease activity. The PCR product (P1) shares 419 and 798 bp overlapping sequences with R1 and T1, respectively. This shows that all the clones (R1, P1, and T1) are derived from a transcript of the same type of Na⁺ channel. These clones collectively encompassed 8,378 bp of cDNA, which was designated nRNaCh. Fig. 1 depicts the positions of these overlapping clones, which constitute nRNaCh.

Fig. 1

The overlapping cDNA clones that constitute nRNaCh. The open box indicates the protein-coding region. Closed boxes represent each cDNA clone. The clone R1 (nt 1-2,686) was isolated from the random-primed library using the probe P_I and the clone T1 (nt 5,068-8,378) was isolated from the oligo(dT)-primed library with the probe P_IV. The solid lines indicate the probes. P_I was obtained by RT-PCR using primers IS and IAS, P_IV was obtained using primers IVS and IVAS. The cDNA corresponding to the remaining portion of nRNaCh between R1 and T1 was isolated by RT-PCR (P1, nt 2,268–5,865) using primers PS and PAS.

The complete nucleotide sequence of nRNaCh consists of a 227 bp 5′-untranslated sequence, an open reading frame of 6,024 bp, and an 2,127 bp 3′-untranslated region. The Met codon at nucleotide residues 228–230 was deemed the translation initiation site, because this is the first in-frame ATG triplet that appears downstream from the nonsense codon TAA (residues 207–209). This assignment is supported by the fact that the sequence around the ATG triplet agrees with the favored sequence for eukaryotic initiation sites, that is A/GXXAUGG, where X can be any nucleotide (Kozak, 1981). The 3′-untranslated region contains a potential polyadenylation signal (AATAAA) 21 bp before the poly(dA) region. The continuous open reading frame of 6,024 bp encodes 2,007 amino acid residues with a calculated molecular weight of 228, 397. Fig. 2 shows the nucleotide sequence and deduced amino acid sequence of nRNaCh. The amino acid sequence of nRNaCh predicts general structural features shared with other cloned Na⁺ channels, 4 homologous repeating units with six putative transmembrane helices in each repeat. In general, the transmembrane segments are well conserved, whereas the putative cytoplasmic and extracellular loops are more divergent and contain a number of insertions or deletions. However, the cytoplasmic loop between S6 of repeat III and S1 of repeat IV(III-IV linker) is well conserved and contain IFM tri-peptide motif (residues 1489–1491) that is essential for fast inactivation. The nRNaCh contains seven potential cAMP-dependent protein kinase (PKA) sites, nine potential N-linked glycosylation sites and one potential protein kinase C phosphorylation site in the III-IV linker (West, 1991). The length of the nRNaCh S5–S6 loop of repeat I is similar to that of TTX-sensitive channels, while there is a deletion in this region of TTX-resistant Na⁺ channels. There is a non-aromatic amino acid (Ala) at the 3^rd position in the SS2 region of repeat I of nRNaCh, the 386^th residue of nRNaCh.

Fig. 2

Nucleotide sequence of the cDNA encoding the putative newt retina sodium channel (above) and its deduced amino acid sequence (below). The complete 8,378 nucleotide sequence has been deposited in the GenBank database with the accession number AF123593. The sequence was determined from the clones R1, P1, and T1 (Fig. 1). The suggested location of six transmembrane helices and SS1-SS2 region within each repeat are designated by horizontal lines (Noda et al., 1986; Guy and Conti, 1990). Predicted intracellular cAMP-dependent protein kinase (PKA) sites are indicated with closed triangles (▴), predicted extracellular N-glycosylation sites are indicated with asterisks (*) and the protein kinase C site involved with modulation of channel activity (West et al., 1991) is indicated with an open triangle (▵).

In situ hybridization analysis of Na⁺ channel expression

We performed in situ hybridization using a digoxigenin-labeled cRNA probe to examine expression of nRNaCh within the retina (Fig. 3). In sections of newt eye, antisense probe distinctly hybridized to the ganglion cell layer throughout the retina, from the central to peripheral regions. It was reported that the ganglion cell layer consists of spiking neurons; ganglion cells and displaced amacrine cells (Ball and Dickson, 1983). Thus, nRNaCh is expressed primarily in ganglion cells and displaced amacrine cells. Weak signals were detected on the vitreal side of the inner nuclear layer, where amacrine cells and displaced ganglion cells are located (Ball and Dickson, 1983). Therefore, nRNaCh is expressed exclusively in spiking neurons throughout the newt retina.

Fig. 3

In situ hybridization showing nRNaCh expression in the newt retina. (A) nRNaCh expression is observed in the ganglion cell layer of the retina using DIG-labeled antisense nRNaCh probe. (B) The control is not stained with the sense probe. Abbreviations: RPE, retinal pigment epithelial cell layer; ONL, outer nuclear cell layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 100 μm.

Comparison with other Na⁺ channels

The sequences of vertebrate Na⁺ channels, for which TTX sensitivity is known, were aligned using the program GENETYX (Software Development Co., Ltd. Japan). Using the UPGMA method, the simple relationship shown in Fig. 4 was obtained from the alignment. The sequences fall into two obvious groups, the TTX-R group, which are the TTX-resistant channels less nRNaCh, and the TTX-S group, which are the TTX-sensitive channels plus nRNaCh. Although nRNaCh is TTX resistant, it is more homologous to channels in the TTX-S group.

Fig. 4

A comparison of vertebrate Na⁺ channel sequences with known TTX sensitivity. The amino acid sequences were aligned using the program GENETYX. The genetic distances between the sequences were estimated using the UPGMA method and are reflected in the length of the horizontal lines. Sequences: hBNaCh, human brain TTX-sensitive channel (Ahmed et al., 1992); rBNaCh2, rat brain type II TTX-sensitive channel (Noda et al., 1986); rBNaCh1, rat brain type I TTX-sensitive channel (Noda et al., 1986); rBNaCh3, rat brain type III TTX-sensitive channel (Kayano et al., 1988); nRNaCh, newt retina TTX-resistant channel; hNNaCh, human neuroendocrine cell TTX-sensitive channel (Klugbauer et al., 1995); rMNaCh1, rat skeletal muscle TTX-sensitive channel (Trimmer et al., 1989); hMNaCh, human skeletal muscle TTX-sensitive channel (George et al., 1992); eENaCh, eel electroplax TTX-sensitive channel (Noda et al., 1984); dGNaCh, dog nodose ganglion neuron TTX-resistant channel (Chen et al., 1997); rSNaCh, rat sensory neuron TTX-resistant channel (Akopian et al., 1996; Sangameswaran et al., 1996); hHNaCh, human heart TTX-resistant channel (Gellens et al., 1992); rHNaCh, rat heart TTX-resistant channel (Rogart et al., 1989; Kallen et al., 1990);. The TTX-resistant channels are in boxes.