A Novel Tetrodotoxin-sensitive, Voltage-gated Sodium Channel Expressed in Rat and Human Dorsal Root Ganglia

Dorsal root ganglion neurons express a wide repertoire of sodium channels with different properties. Here, we report the cloning from rat, dorsal root ganglia (DRG), cellular expression, and functional analysis of a novel tetrodotoxin-sensitive peripheral sodium channel (PN), PN1. PN1 mRNA is expressed in many different tissues. Within the rat DRG, both the mRNA and PN1-like immunoreactivity are present in small and large neurons. The abundance of sodium channel mRNAs in rat DRG is rBI > PN1 ≥ PN3 >>> rBIII by quantitative reverse transcription-polymerase chain reaction analysis. Data from reverse transcription-polymerase chain reaction and sequence analyses of human DRG and other human tissues suggest that rat PN1 is an ortholog of the human neuroendocrine channel. In Xenopus oocytes, PN1 exhibits kinetics that are similar to rBIIa sodium currents and is inhibited by tetrodotoxin with an IC50 of 4.3 ± 0.92 nm. Unlike rBIIa, the inactivation kinetics of PN1 are not accelerated by the coexpression of the β-subunits. Dorsal root ganglion neurons express a wide repertoire of sodium channels with different properties. Here, we report the cloning from rat, dorsal root ganglia (DRG), cellular expression, and functional analysis of a novel tetrodotoxin-sensitive peripheral sodium channel (PN), PN1. PN1 mRNA is expressed in many different tissues. Within the rat DRG, both the mRNA and PN1-like immunoreactivity are present in small and large neurons. The abundance of sodium channel mRNAs in rat DRG is rBI > PN1 ≥ PN3 >>> rBIII by quantitative reverse transcription-polymerase chain reaction analysis. Data from reverse transcription-polymerase chain reaction and sequence analyses of human DRG and other human tissues suggest that rat PN1 is an ortholog of the human neuroendocrine channel. In Xenopus oocytes, PN1 exhibits kinetics that are similar to rBIIa sodium currents and is inhibited by tetrodotoxin with an IC50 of 4.3 ± 0.92 nm. Unlike rBIIa, the inactivation kinetics of PN1 are not accelerated by the coexpression of the β-subunits. Voltage-gated sodium channels play a critical role in the rising phase of action potential and are, thus, important for impulse generation and conduction in most excitable cells. Sodium channels are integral membrane proteins that are usually comprised of one large α-subunit (>200 kDa) and one or more smaller β-subunits (1Catterall W.A. Physiol. Rev. 1992; 72: S15-S48Google Scholar, 2Isom L.L. De Jongh K.S. Catterall W.C. Neuron. 1994; 12: 1183-1194Google Scholar). Several α-subunit sodium channel genes have been isolated from different tissues and functionally analyzed in heterologous expression systems (3Catterall W.A. Curr. Opin. Neurobiol. 1991; 1: 5-13Google Scholar). We and others have previously isolated clones from a dorsal root ganglia (DRG) 1The abbreviations used are: DRGdorsal root gangliaPNperipheral sodium channelTTXtetrodotoxinhNEhuman neuroendocrine channelRTreverse transcriptionPCRpolymerase chain reactionQRT-PCRquantitative RT-PCRbpbase pairPBSphosphate-buffered salineTEVCtwo-electrode voltage clampkbkilobase(s) cDNA library for a novel tetrodotoxin-resistant sodium channel, PN3/SNS, which is expressed only in sensory neurons (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar, 5Akopian A.N. Sivilotti L. Wood J. Nature. 1996; 379: 257-262Google Scholar). In addition to the PN3 cDNA clones, we have isolated cDNAs for other known and novel sodium channels (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar). One of them, PN1 (peripheral sodium channel 1) (PN1), is a sodium channel that is regulated by nerve growth factor in PC12 cells (6D'Arcangelo G. Paradiso K. Sheperd D. Brehm P. Halegoua S. Mandel G. J. Cell. Biol. 1993; 122: 915-921Google Scholar, 7Toledo-Aral J.J. Brehm P. Halegoua S. Mandel G. Neuron. 1995; 14: 607-611Google Scholar). Nerve growth factor increases the mRNA expression levels of brain type II/IIa and PN1 by two distinct signaling mechanisms (7Toledo-Aral J.J. Brehm P. Halegoua S. Mandel G. Neuron. 1995; 14: 607-611Google Scholar). To further understand the molecular basis of neuronal excitability, we now describe the isolation of full-length cDNA clones for PN1, quantitation of PN1 mRNA levels as compared with other sodium channels in DRG, tissue distribution and its cellular localization in DRG, and functional characterization of this TTX-sensitive sodium channel in Xenopus oocytes. In addition, we discuss the isolation of partial clones of the human ortholog of PN1 (human neuroendocrine channel (hNE)) and the distribution of hPN1/NE sodium channel by RT-PCR analysis. Our studies indicate that PN1 is a novel TTX-sensitive sodium channel that is highly homologous to the rabbit sodium channel Nas (8Belcher S.M. Zerillo C.A. Levinson R. Ritchie J.M. Howe J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11034-11038Google Scholar) and expressed in a wide variety of tissues, including all cell types within the DRG, and that the human PN1 ortholog is, indeed, the neuroendocrine channel, hNE (9Klugbauer N. Lacinova L. Flockerzi V. Hoffmann F. EMBO J. 1995; 14: 1084-1090Google Scholar). dorsal root ganglia peripheral sodium channel tetrodotoxin human neuroendocrine channel reverse transcription polymerase chain reaction quantitative RT-PCR base pair phosphate-buffered saline two-electrode voltage clamp kilobase(s) Two rat DRG cDNA libraries, one oligo(dT) and the other random hexamer-primed, were constructed and screened as described (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar). The cDNA probe for screening the random-primed cDNA library was a cDNA fragment from domains I and II of an rBIIa clone (10Auld V.J. Goldin A.L. Krafte D.S. Marshall J. Dunn J.M. Catterall W.A. Lester H.A. Davidson N. Dunn R.J. Neuron. 1988; 1: 449-461Google Scholar). Overlapping cDNA clones 27.6, 62.5, and 69.1 were assembled in the Xenopusoocyte expression vector, pBSTA (16Goldin A.L. Sumikawa K. Methods Enzymol. 1992; 207: 279-297Google Scholar), at theAvrII/BspEI sites. The overlapping cDNA clones 27.6, 62.5, 63.1, and 69.1, the resulting clones at various stages of assembly, and the final pBSTA-PN1 clone were sequenced on both strands. Sequence analyses were performed as described previously (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar). Human tissues were provided by the Department of Pathology, Stanford University, Palo Alto, CA. Total RNA from human adrenal, heart, and brain tissues, and poly(A)+ RNA from thyroid tissue were purchased fromCLONTECH (Palo Alto, CA). Extraction of total RNA and synthesis of first strand cDNA from rat and human tissues were as described earlier (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar). Tissue distribution analysis and quantitative RT-PCR (QRT-PCR) were performed with primer sets within the 3′-noncoding region of the gene. The forward primers targeted the same sequence, whereas the reverse primer for QRT-PCR was more upstream in the noncoding region. The amplicon sizes for tissue distribution analysis and the QRT-PCR experiment were 646 and 180 bp, respectively. Thermal cycler parameters were 30 s at 94 °C, 30 s at 61 °C, 1 min at 72 °C (34 cycles) and 30 s at 94 °C, 30 s at 61 °C, 5 min at 72 °C (1 cycle). For QRT-PCR, 100 ng total RNA were reverse transcribed to single-stranded cDNA, and the PCR was performed in the presence of 1 μCi 32PdCTP and the following parameters: 30 s at 94 °C, 30 s at 55 °C and 2 min at 72 °C (35 cycles). Specific primers targeting the 3′-noncoding region for PN3, rBI, and rBIII were similarly employed for QRT-PCR. An aliquot of the reactions was run on a polyacrylamide gel that was dried prior to quantitation on a PhosphoImager (Bio-Rad). An external control cRNA comprising the same fragment was constructed for QRT-PCR as described by Ramakrishnan et al. (11Ramakrishnan R. Fink D.J. Jiang G. Desai P. Glorioso J.C. Levine M. J. Virol. 1994; 68: 1864-1873Google Scholar). A standard curve was generated with the external controls for every experiment. 1 ng of pBK-PN1 plasmid DNA was included as positive control for the tissue distribution analysis by RT-PCR. Primers for glyceraldehyde-3-phosphate dehydrogenase were employed to demonstrate tissue viability (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar) and to normalize the RNA content in QRT-PCR experiments. Nested, degenerate primers from cloned human sodium channel sequences (12Ahmed C.M.I. Ware D.H. Lee S.C. Patten C.D. Ferrer-Montiel A.V. Schinder A.F. McPherson J.D. Wagner-McPherson C.B. Wasmuth J.J. Evans G.A. Montal M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8220-8224Google Scholar, 13Gellens M.E. George A.L. Chen L. Chahine M. Horn R. Barchi R. Kallen R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 554-558Google Scholar, 14George A.L. Komisarof J. Kallen R.G. Barchi R.L. Ann. Neurol. 1992; 31: 131-137Google Scholar, 15George A.L. Knittle T.J. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4893-4897Google Scholar) were used to amplify a 687-bp fragment spanning domain IVS1 to IVS6. Thermal cycler parameters were: first set of primers, 1 min at 94 °C, 2 min at 50 °C, 4 min at 72 °C (34 cycles) and 1 min at 94 °C, 2 min at 50 °C, 8 min at 72 °C (1 cycle); and for the second set of primers, 1 min at 94 °C, 2 min at 55 °C, 4 min at 72 °C (34 cycles) and 1 min at 94 °C, 2 min at 55 °C, 8 min at 72 °C (1 cycle). Specific primers for interdomain I/II and interdomain II/III based hNE channel sequence (9Klugbauer N. Lacinova L. Flockerzi V. Hoffmann F. EMBO J. 1995; 14: 1084-1090Google Scholar) defined amplicon sizes of 813 and 352 bp, respectively. Thermal cycler parameters were: 30 s at 94 °C, 30 s at 60 °C, 2 min at 72 °C (35 cycles) and 30 s at 94 °C, 30 s at 60 °C, 7 min at 72 °C (1 cycle). Blots were prepared from the agarose gels that were used to fractionate the PCR fragments and hybridized with a32P-labeled DNA probe representing the entire coding sequence of the hNE channel (9Klugbauer N. Lacinova L. Flockerzi V. Hoffmann F. EMBO J. 1995; 14: 1084-1090Google Scholar). Rats (125–150 g, Fisher and Charles River) were anesthetized with 10% chloral hydrate and perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (pH 7.6). DRG (L4–5) were dissected out, cryoprotected by incubation in PBS-sucrose (10–20%), then frozen and sectioned (10 μm) with a cryostat. Sections were thawed, digested in proteinase K (1 μg/ml) for 1 h at 37 °C, dehydrated in ethanol (50–100%), and air dried. Specific activity of the oligonucleotide probes (targeted for the 3′-untranslated region of PN1 mRNA) was 5 × 107 cpm/μg. Hybridization, washing conditions, and development of sections were identical to those used for PN3 in situ hybridization (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar). Selected sections were counterstained with propidium iodide. To perform in situ hybridization using non-radioactive oligonucleotide probes, the following protocol was used. After dewaxing/deparaffinization series in xylene and ethanol, 4-μm sections were permeabilized with Triton X-100 (0.3%, 15 min, RT) and subsequently with HCl (0.2 m), pepsin (0.1%, 10 min, 37 °C), and proteinase K (10 mg/ml, 30 min, 37 °C). Following the permeabilization, sections were postfixed in 4% formaldehyde, washed in PBS, and acetylated on a shaking platform with acetic anhydride (0.25%, in triethanolamine buffer, pH 8). Sections were pre-hybridized for 2 h at 37 °C in a hybridization solution containing 2 × SSC buffer, 1 × Denhardt's solution, 10% Dextran sulfate, 250 mg/ml yeast tRNA, 0.05 pm/ml of Randomer Oligoprobe (DuPont), 0.1 mg/ml poly(A) (Boehringer Mannheim), 500 mg/ml of denatured salmon testis DNA, and 50% deionized formamide. Hybridization was done in the same solution, containing specific antisense or sense oligoprobe, overnight at 37 °C. Following hybridization, samples were washed twice with 2, 1, and 0.25 × SSC, for 15 min at 37 °C each, followed by a rinse in buffer A (100 mm Tris-HCl (pH 7.5) plus 150 mmNaCl). Next, samples were incubated in blocking solution (buffer A plus 0.1% Triton X-100 plus 150 mm NaCl, 1 h, RT) and then in anti-digoxigenin antibodies conjugated with alkaline phospatase (2 h, RT). Samples were subsequently washed in buffer A, incubated in detection buffer (100 mm Tris-HCl plus 100 mmNaCl plus 50 mm MgCl2), and then in 200 mm nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate plus 10 mm of levamisol in 10 ml of detection buffer A (Boehringer Mannheim detection kit) overnight in the dark. Next day, sections were washed with water and mounted with Krystalon. Three peptides, two 16-mers and one 14-mer (located in interdomain I/II, II/III and domain IVS1/2), were synthesized according to the multiple antigen peptide technology. Polyclonal antisera were raised in rabbits (Research Genetics, AL) and affinity purified. The antipeptide antibody raised against the 16-amino acid peptide (1035–1050 amino acid in the protein sequence) located in the extracellular loop between domain IVS1 and S2 was employed in the immunocytochemical localization of PN1 (see below). Male Sprague-Dawley rats (200–250 g; Harlan, Indianapolis, IN) were anesthetized with 10% chloral hydrate and perfused with PBS followed by 10% formalin. Brain, DRG (L4–5), and lumbar spinal cord were removed and postfixed overnight in 10% formalin. Following fixation, tissue was embedded in paraffin and sectioned at 4 μm. After deparaffinization, sections were preincubated in PBS containing 20% normal goat serum and 0.2% Triton X-100 and incubated with PN1 antibodies (1/1000, overnight, at 4 °C). Tissue sections were washed, incubated in biotinylated anti-rabbit IgG (1/200, RT; Vectastain Elite) followed by peroxidase avidin-biotin complex (1/50, 90 min, RT; ABC, Vectastain Elite), and visualized with a diaminobenzidene reaction. Finally, the tissue sections were washed again, dehydrated, and coverslipped for light microscopy For the control antibodies were with the A was used for following both in situ hybridization and cRNA was synthesized from PN1 pBSTA (16Goldin A.L. Sumikawa K. Methods Enzymol. 1992; 207: 279-297Google Scholar) using an and were prepared A.L. Methods Enzymol. 1992; 207: Scholar) and with PN1 cRNA using a For two-electrode voltage clamp that been with of cRNA for previously were perfused with a solution containing NaCl, 2 1 and (pH with Lester H.A. N. J. Physiol. 1994; Scholar), and voltage with a 500 in were at and to different for 15 For that been with 10 ng of PN1 cRNA previously were R.G. Barchi R.L. Neurobiol. 1993; Scholar) in plus 100 mm The solution NaCl, 1 1 and 5 (pH The was to the an agarose agarose in were from or on a and with A.L. Methods Enzymol. 1992; 207: Scholar), which NaCl, 2 1 and 5 (pH were with and were from using a in clamp To the was using the were not for the were at and were to different for For both and and were by a Data was at 50 at 5 or 10 with a For or F. J. Physiol. Scholar) was used. For was 5 with inactivation used to and were by the which was from the at and The sequence of a clone from the RT-PCR analysis of rat DRG using degenerate primers, to rat sodium channel domain a novel sodium channel. this was to identical to of the sequence of clone 27.6, isolated from the cDNA sequence analysis of the full-length cDNA assembled from clones 27.6, 62.5, and 69.1 a pair coding for a acid protein In addition, clone a 3′-noncoding sequence, and clone a pair upstream The assembled clone comprised of the in (see was an upstream of the to other cloned sodium channels (10Auld V.J. Goldin A.L. Krafte D.S. Marshall J. Dunn J.M. Catterall W.A. Lester H.A. Davidson N. Dunn R.J. Neuron. 1988; 1: 449-461Google Scholar). the of the sodium channels were in the amino acid sequence of PN1, including for protein A. 5 and in domain is the amino that to of (see R.G. Barchi R.L. Neurobiol. 1993; Scholar). PN1 with the human channel and rabbit sodium channel rBIII and rBI PN1 to the brain sodium rBI, and rBIII in 2 L. 1996; Scholar). A of the sodium channel in the human DRG was performed by RT-PCR with degenerate primers from domain The sequence of one of the clones from this region was identical to the hNE channel not 8). analyses with a cDNA probe from an that was in DRG and to a in brain not RT-PCR analyses that PN1 mRNA was expressed in a wide variety of rat tissues, The mRNA levels of PN1, PN3, rBI, and rBIII in the rat DRG by QRT-PCR are in that PN1 and PN3 mRNAs were expressed at rBI mRNA was to more PN1 and PN3, and rBIII was the of distribution of rat PN1 and human channel mRNAs by RT-PCR not not in a of sodium channel mRNAs in rat DRG by QRT-PCR channel RNA ± ± ± ± ± in a from a variety of human tissues were used to perform RT-PCR with specific primers for hNE I/II and and the PCR were by sequence analysis and hybridization The in that the hNE channel mRNA was expressed in the same tissues as rat PN1, including and thyroid tissues as was described by et al. (9Klugbauer N. Lacinova L. Flockerzi V. Hoffmann F. EMBO J. 1995; 14: 1084-1090Google Scholar). hNE rat PN1 was expressed in The sequences of the PCR fragments spanning I/II and II/III from DRG were identical to the hNE channel. of sequences spanning many of the coding sequence of hNE in human DRG that the channel was expressed in this the in mRNA sizes of rat PN1 and (9Klugbauer N. Lacinova L. Flockerzi V. Hoffmann F. EMBO J. 1995; 14: 1084-1090Google Scholar) have to is that rat PN1 a 3′-untranslated region The sequence between rat PN1 and with the that hNE is the human ortholog of the rat PN1 and rabbit sodium channels (8Belcher S.M. Zerillo C.A. Levinson R. Ritchie J.M. Howe J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11034-11038Google Scholar). PN1 mRNA is expressed in small and large DRG as by in situ hybridization in A. 2 the same with propidium to the and of neurons. were with in situ hybridization 2 In with the in situ hybridization with PN1 antipeptide antibody that all DRG neurons with In control the antibody with the peptide was was immunoreactivity the of the In addition, a distribution of PN1 immunoreactivity was in rat spinal cord and brain not of DRG neurons. A, with PN1 antibody that all neurons with paraffin × two neurons that are of similar with different antibody DRG was incubated with the PN1 antibody that been with the peptide antigen with PN1 cRNA that expression of PN1 an that compared with TTX-sensitive sodium currents in and DRG J.R. J. 1988; Scholar, J. Physiol. Scholar, J.R. J. Physiol. 1993; Scholar). The for using was ± ± of the rat is known to the inactivation kinetics of the and rat sodium channels using by a not of PN1 cRNA with 5 ng of L.L. De Jongh K.S. J. K. Goldin A.L. Catterall W.A. 1992; Scholar) 1 ng of L.L. D.S. De Jongh K.S. Catterall W.A. Cell. 1995; Scholar) or a of and not to the inactivation kinetics not to the large of Xenopus the of is To PN1 currents with more we from of levels of PN1 In for 4 was to ± ± is similar to the voltage of of A. J. Physiol. 1994; Scholar) and J. Neuron. 1991; Scholar) using this with the the currents and more The inactivation was with of the the with of inactivation were with of and at and 0.1 and at are with the that this channel exhibits more one as been for other sodium channels L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; Scholar, A. J. Physiol. 1994; Scholar, J. Neuron. 1991; Scholar, J. 1988; Scholar, J.R. Neuron. Scholar). of inactivation using and 10 s 4 a of ± ± voltage is to that of using this protocol ± more that of ± The PN1 was by with an IC50 of 4.3 ± 0.92 that PN1 is a TTX-sensitive sodium channel, with similar to that of the Goldin A.L. Neuron. 1991; Scholar), M.E. George A.L. Chen L. Chahine M. Horn R. Barchi R.L. Kallen R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 554-558Google Scholar), rBIII J. S.M. Kallen R.G. Barchi R.L. Mandel G. Neuron. S. N. M. S. 1988; Scholar), and hNE (8Belcher S.M. Zerillo C.A. Levinson R. Ritchie J.M. Howe J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11034-11038Google Scholar) channels with the J.R. G. Scholar, L.L. J. H.A. Scholar) and (4Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Google Scholar, 5Akopian A.N. Sivilotti L. Wood J. Nature. 1996; 379: 257-262Google Scholar) sodium In PN1 is a novel sodium channel that is not to previously sodium as is to of in to rBIIa, using PN1 exhibits inactivation kinetics that are not accelerated by coexpression of the β-subunits. In DRG, PN1 to expressed at levels that are to those of Unlike PN3, PN1 is expressed in all types of neurons in In of sequence we that rat PN1, and rabbit are of the same sodium channel gene. The wide distribution of that to in types of neurons and excitable cells. We P. and for sequence analysis and oligonucleotide synthesis and for on PN1 We are to A. for and from rBIIa and We are to R. L. for and the of this