Molecular cloning and functional expression of the human sodium channel b 1B subunit, a novel splicing variant of the b 1 subunit Ning Qin 1 , Michael R. D’Andrea 1 , Mary-Lou Lubin 1 , Navid Shafaee 2, *, Ellen E. Codd 1 and Ana M. Correa 2 1 Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA, USA; 2 Department of Anesthesiology, University of California, Los Angeles, CA, USA The voltage gated sodium channel comprises a pore-forming a subunit and regulatory b subunits. We report here the identification and characterization of a novel splicing variant of the human b 1 subunit, termed b 1B . The 807 bp open reading frame of the human b 1B subunit encodes a 268 residue protein with a calculated molecular mass of 30.4 kDa. The novel human b 1B subunit shares an identical N-terminal half (residues 1–149) with the human b 1 subunit, but contains a novel C-terminal half (residues 150–268) of less than 17% sequence identity with the human b 1 subunit. The C-terminal region of the human b 1B is also significantly different from that of the rat b 1A subunit, sharing less than 33% sequence identity. Tissue distribution studies reveal that the human b 1B subunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle. Functional studies in oocytes demonstrate that the human b 1B subunit increases the ionic current when coex- pressed with the tetrodotoxin sensitive channel, Na V 1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human b 1 and rat b 1A subunits. Keywords: sodium channel; b 1B subunit; splicing variant. By mediating the rapid entry of sodium ions into excitable cells in response to voltage changes across the plasma membrane, voltage gated sodium channels (VGSCs) play a fundamental role in the control of neuronal excitability in the central and peripheral nervous systems. The VGSC is a heteromeric protein complex that comprises at least a large (200–300 kDa) pore-forming a subunit and several smaller (30–40 kDa) regulatory b subunits [1–4]. It is well known that sodium channel a subunits determine the basic properties of the channel, while b subunits modulate the channel properties. Functional studies in a heterologous system have demonstrated that, depending on the type of coexpressed a subunit, b subunits are able to modulate almost all aspects of the channel properties, including voltage dependent gating, activation and inactivation, as well as greatly increasing the number of functional channels present on the plasma membrane [5,6]. Currently, at least nine different a subunits, three b subunits, and a splicing variant of the b 1 subunit, rat b 1A [7], have been cloned and characterized. The rat b 1A subunit is a splicing variant of the b 1 subunit via intron retention. The N-terminal half of the b 1A subunit is identical to that of the rat b 1 subunit, whereas its C-terminal half, encoded by a retained intron with an in-frame stop codon, is completely different from that of the rat b 1 subunit (to which it shows less than 17% identity). Coexpression of the rat b 1A subunit with the pore forming alpha subunit, Na V 1.2, in Chinese hamster lung 1610 cells, increased the sodium current density and produced subtle changes in voltage depend- ent activation and inactivation [7]. To further explore the function and physiological relevance of the sodium channel b 1 splicing variant, we first tried to clone the same splicing variant from human tissue. Here, we report the cloning and characterization of a novel, splicing variant of the human b 1 subunit by rapid amplification of cDNA end polymerase chain reaction (RACE-PCR) based on the human b 1 sequence. The novel b 1 subunit splicing variant, named b 1B , is produced via extension of exon 3 with an in-frame stop codon. The human b 1B subunit is significantly different from the rat b 1A subunit in sequence, expression pattern and regulatory properties, although they share a similar splicing pattern. Functional studies indicate that the human b 1B subunit performs a physiological function distinct from that of the human b 1 subunit when it is coexpressed with Na V 1.2 in Xenopus oocytes. Experimental procedures Molecular cloning of the human sodium channel b 1B subunit Full-length human b 1B cDNA was cloned using a strat- egy that combined reverse transcription polymerase chain Correspondence to N. Qin, Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, PO Box 776, Welsh and McKean Roads, Spring House, PA 19477-0776, USA. Fax: + 1 215 628 3297, Tel.: + 1 215 540 4886, E-mail: nqin@prdus.jnj.com Abbreviations: DRG, dorsal root ganglia; RACE-PCR, rapid ampli- fication of cDNA end-polymerase chain reaction; RT-PCR, reverse transcription–polymerase chain reaction; VGSC, voltage gated sodium channel. *Present address: Royal College of Surgeons, Dublin, Ireland. (Received 28 July 2003, revised 6 October 2003, accepted 15 October 2003) Eur. J. Biochem. 270, 4762–4770 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03878.x reaction (RT-PCR) and RACE-PCR. Marathon-Ready TM human adrenal gland and fetal brain cDNA libraries were purchased from Clontech (Palo Alto, CA, USA). The RACE-PCR was performed according to the supplier’s instructions. The reaction mixture (50 lL final volume) contained 5 lL of Marathon-Ready TM human adrenal gland cDNA, 200 l M dNTP, 200 n M AP1 primer (Clon- tech), 200 n M human b 1 subunit specific primer (SB1-10: 5¢-TGGACCTTCCGCCAGAAGGGCACTG-3¢), and 1 lLof50· Advatage2 DNA polymerase mixture (Clon- tech). The thermal cycling parameters for RACE–PCR were as follows: an initial denaturation at 94 °C for 30 s; five cycles of 94 °Cfor5sand72°C for 4 min; five cycles of 94 °Cfor 5sand70°C for 4 min; and 20 cycles of 94 °Cfor5sand 68 °C for 4 min. The RACE–PCR product was then cloned into the PCR-Script TM Amp Cloning vector (Statagene, La Jolla, CA, USA), according to the protocol provided by the supplier. The full-length b 1B subunit was cloned from the Mara- thon-Ready TM human fetal brain cDNA library based on the C-terminal sequence of human b 1B subunit obtained using the RACE-PCR. The PCR was performed in a final volume of 50 lL, containing 5 lL of Marathon-Ready TM humanfetalbraincDNA,5lLof10· reaction buffer, 200 l M dNTP, 200 n M SB1-6 primer (5¢-GCCATGGG GAGGCTGCTGGCCTTAGTGGTC-3¢) and SB1-19 pri- mer (5¢-GTGTGCCTGCAGCTGCTCAA-3¢), and 1 lL of 50· HF2 DNA polymerase mixture (Clontech). Four independent clones were selected and subjected to double stranded DNA sequencing analysis. All four independent clones from the human fetal brain were found to contain sequences identical to that of the RACE-PCR cloned b 1B subunit from human adrenal gland. Generation of polyclonal antibody A peptide (RWRDRWQAVDRTGC), derived from the C terminus of the human b 1B subunit, was synthesized and used for raising polyclonal antibodies in rabbits. (This peptide was chosen because the seqeunce shows the highest homology between human and rat b 1A subunits.) The antibody was raised and affinity purified by BioSource International, Inc. The resulting affinity purified antibody was used for immunohistochemical analysis. Northern blot analysis Human Multiple Tissue Northern blot (MTN TM )and human Brain II MTN TM blot were purchased from Clontech. The cDNA fragment encoding residues 217– 268 of the human b 1B subunit was used as a probe. The antisense single stranded DNA probe was synthesized using the Strip-EZ TM PCR kit (Ambion, Austin, TX, USA), in the presence of antisense primer SB1-20 (5¢-TC AAACCACACCCCGAGAAA-3¢)and[ 32 P]dATP[aP] (3000 CiÆmmol )1 ; Amersham Pharmacia Biotech.), follow- ing the manufacturer’s instructions. The labeled probe was then separated from free [ 32 P]dATP[aP] using a Micro- Spin TM G-50 column (Amersham Pharmacia Biotech.). The cDNA fragment encoding the human b 1 subunit from amino acids 150 to 218 was used as a human b 1 subunit specific probe. The single stranded antisense b 1 specific probe was labeled and purified as described above. A 2 kb human b-actin cDNA fragment was used as the control probe and labeled with Ready-To-Go TM DNA Labelling Bead (–dCTP) (Amersham Pharmacia Biotech.), followed by purification as described above. The blots were prehybridized with 5 mL of UltraHyb Solution (Ambion), at 42 °C for 2 h, and then hybridized in the presence of 1 · 10 6 c.p.m.ÆmL )1 probe (b 1B , b 1 and b-actin separately) at 42 °C overnight. The blots were washed with 2 · 200 mL of 0.2· NaCl/Cit/0.1% SDS, at 65 °C for 2 h. Finally, the blots were exposed to X-ray film in a )80 °C freezer for 2–18 h. The same blots were used for all three probes (b 1B , b 1 and b-actin) after stripping at 68 °C for 15 min and reconstitution at room temperature for 15 min using the Strip-EZ TM removal kit (Ambion). Immunohistochemistry Protocols for immunohistochemistry have been described previously [8]. All incubations were performed at room temperature. After microwaving the slides in Target (Dako, Carpenturia, CA, USA), the slides were placed in NaCl/P i andthenin3%H 2 O 2 ,rinsedinNaCl/P i and then the appropriate blocking serum was added for 10 min. Subsequently, primary antibody, rabbit polyclonal anti-(human b 1B ), at a titer of 1 : 200, was applied to the slides for 30 min. After several washes in NaCl/P i ,a biotinylated secondary antibody (Vector Laboratories) was placed on the slides for 30 min. Subsequently, the slides were washed in NaCl/P i and the avidin–biotin complex (ABC; Vector Laboratories) was applied to the cells for 30 min. The presence of the primary antibody was detected after two 5 min incubations in 3¢-diaminobenzi- dine-HCl (Biomeda, Foster City, CA, USA). Slides were briefly exposed to Mayer’s hematoxylin for 1 min, dehy- drated and coverslipped. Antibody specificity controls included (a) replacement of the primary antibody with nonimmune serum, (b) omission of the primary antibody with the antibody dilution buffer (Zymed Laboratories Inc., San Francisco, CA, USA), and (c) preincubation with specific antigen (preabsorption). Preabsorption was carried out using a 10-fold titer excess of the antigen peptide preincubated with antibody overnight at 4 °C. This mixture was then used as the Ôprimary antibodyÕ. b 1B subunit immunolabeling was not detected in the preab- sorption controls or in other negative controls. Specimens were examined and photographed using an Olympus BX-50 microscope. In vitro synthesis of cRNA The expression constructs of b 1B and Na V 1.2 were linearized following digestion with restriction enzymes. The cRNAs were synthesized in vitro with T7 RNA polymerase using reagents and protocols from the mMESSAGE mMACHINE TM transcription kit (Ambion), except that the LiCl precipitation was repeated twice. To ensure full- length clones of the Na V 1.2 a subunit, the reaction mixture was supplemented, halfway through transcription, with additional enzyme and nucleotides. The cRNAs were Ó FEBS 2003 Cloning and characterization of Na + channel b 1B (Eur. J. Biochem. 270) 4763 suspended in diethylpyrocarbonate-treated H 2 O at a final concentration of 1–2 mgÆmL )1 . Oocyte preparation and RNA injection Conventional methods were followed for oocyte isolation and removal of the follicular membrane [9]. Adult female Xenopus laevis (Xenopus One, Ltd, Dexter, MI, USA) were anesthetized by immersion in 0.1% tricaine. Ovaries were removed through an abdominal incision. Ovarian sacs were rinsed in Ca 2+ -free medium and teased apart to expose the oocytes. The follicular layer was removed by treat- ment with collagenase (200 UÆmL )1 ;Gibco)inCa 2+ -free medium, followed by rinsing and storage in saline medium containing Ca 2+ and 50 lgÆmL )1 gentamicin. Stage V–VI oocytes were separated for injection the following day. Normally, 20–25 oocytes per RNA sample were micro- injected, each with 50 nL of 1 mgÆmL )1 cRNA. Combina- tion of subunits was obtained by injecting the premixed cRNAs. Microinjection was performed under sterile, RNase-free conditions. After injection, oocytes were maintained at 18 °C. Recording solutions External and internal recording solutions contained mostly impermeant anions and were made iso-osmolar to the oocyte media (120 m M ; 220–240 mOsmÆkg )1 ). Sodium currents were recorded in external 120 m M sodium methane sulfonate, 1.8 m M CaCl 2 ,10m M Hepes-sodium, pH 7.2; and internal 120 m M cesium methane sulfonate, 10 m M sodium methane sulfonate, 10 m M Hepes-sodium, 1 m M EGTA-sodium, pH 7.2. Voltage electrodes were filled with 2.7 M TMA, which comprised 2.7 M tetra methyl-ammo- nium, 10 m M NaCl, and 10 m M Hepes-sodium, pH 7.0. Recording and analysis of macroscopic ionic and gating currents The cut open oocyte Vaseline gap technique [10] was used to record macroscopic ionic currents. This technique, described previously [11], greatly improves the temporal resolution over that of the conventional two-electrode voltage clamp. The currents were recorded from an area of the oocyte equivalent to 20–25% of the total surface. Voltage electrodes had resistances of 0.2–0.5 MW.Custom- made software and hardware were used for acquisition and analysis of data. Leakage and linear capacity currents were subtracted by using P/4 protocols. Data were sampled once every 5 ls and were filtered at 1/5 of the sampling frequency. Conventional pulse protocols were used to record mac- roscopic sodium currents in response to changes in mem- brane voltage. Test pulses of 15 ms were applied from holding potentials of )80 or )100 mV; the range of test potentials used to cover the whole activation curve was typically )60 mV to 100 mV, at 5 mV intervals. For steady- state inactivation curves, a 15 ms test pulse to 0 mV was preceded by a preconditioning 100 ms pulse spanning )140 mV to 20 mV, at intervals of 10 mV. Conductance vs. voltage (G-V) curves were obtained from the I-V plots fitting the data to: I ¼ G(V)Æ(V m –Vrev), where I is the current amplitude, G(V) is the voltage-dependent conduct- ance, V m is the membrane voltage, and V rev is the voltage for current reversal. Once V rev was determined from the I-V fits, the individual I-V curves were divided by V m –V rev to obtain the G(V). The G-V plots were fitted to: G ¼ G max / (1 + exp[–zÆ(V-V ½ )/25]), where G max is the maximum conductance, z is the valence of the process and V ½ is the midpoint voltage of activation. Ionic current expression levels were determined from batches of oocytes injected with Na V 1.2 alone or with Na V 1.2 combined with b 1B subunit at an a: b ratio of 1 : 5 or 1 : 20. Only data from batches expressing all three a : b combinations were included in the analysis. Unpaired t-test statistics were used to compare the different current amplitude data sets. Results Cloning and analysis of the human VGSC b 1B subunit In order to clone the human b 1 splicing variant, we first used RT-PCR with a forward primer based on the human b 1 subunit and degenerated reverse primers based on the rat b 1A C-terminal sequence. However, these attempts failed to clone the human b 1A subunit from cDNA libraries of adrenal gland, brain and fetal brain. BLAST searches of the human genomic sequence with the cDNA encoding the rat b 1A subunit C terminus also failed to identify any homologous region in the human b 1 gene. These results suggested that, if the human b 1 gene also undergoes alternative splicing, it might have a very different sequence from that of the rat b 1A subunit. Therefore, a RACE-PCR technique was applied to clone a novel, splicing variant of the human b 1 subunit. To perform RACE-PCR of a novel C terminus of the human b 1 subunit, we designed a forward primer (SB1-10) based on the N-terminal cDNA sequence. The resulting RACE-PCR product was directly cloned into the PCR cloning vector. As the human b 1 subunit specific primer was used for 3¢ extension, both the b 1 subunit and its splicing variant would be amplified and cloned. To exclude the b 1 subunit clones, each individual clone was characterized by PCR using a pair of primers for specific amplification of the b 1 subunit C terminus. All PCR negative, non-b 1 subunit clones were subjected to further sequencing analysis, which revealed that one of the clones had a continuous reading frame from the N-terminal sequence of the human b 1 . However, the C-terminal sequence was significantly different from that of the human b 1 , suggesting that it might encode a novel splice variant of the human b 1 subunit. BLAST searches of the National Institutes of Health (NIH) database with this sequence also identified a shorter, but identical, unannotated EST clone (accession number: AI742310) in the human EST database, which was cloned from a pool of five normalized cDNA libraries. Based on the novel C-terminal sequence of the human b 1 subunit obtained by RACE-PCR, a full-length splice variant was cloned from the human fetal brain cDNA library. The full-length cDNA contained a 979 bp sequence encoding 268 amino acids and a 172 bp 3¢ untranslated region (GenBank accession number: AY391842). The amino acid sequence deduced from the cDNA sequence is 4764 N. Qin et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 1. Sequence analysis and genomic struc- ture of the human sodium channel b 1B subunit. (A) Amino acid sequence comparisons between human b 1B and b 1 subunits. The signal peptide sequence and transmembrane domain (TM) are indicated in (A), and an IgG-like motif is located between residues 22 and 150. (B) C-terminal amino acid sequence comparisons of human b 1B, rat b 1A and putative mouse b 1A , which is predicated based on the mouse genomic sequence. Conserved residues are underlined. (C) Genomic struc- ture of the human b 1 gene, SCN1B.The SCN1B gene spans  9kbonchromosome19 across six exons. Exon 3A is an extended exon 3(retentionofpartofintron3)viaalternative splicing. Exons 1, 2, 3, 4 and 5 (solid boxes) encode the b 1 subunit, while exons 1, 2 and 3A (solid and diagonally shaded boxes) encode the b 1B subunit. The 5¢ and 3¢ untranslated regions are indicated by solid thin lines (b 1 ) andshadedthinlines(b 1B ), and the unidenti- fied 3¢ untranslated region of b 1B is indicated using the thin interrupted broken line. The stop codon is indicated by an asterisk. The RACE-PCR primer, SB1-10 (indicated by an arrow), is located at the end of exon 2. Table 1. Intron–exon boundary sequence of the sodium channel b 1 /b 1B gene (SCN1B). UT, untranslated. Exon (bp) cDNA location Codon Acceptor Donor Intron (kb) Exon 1 (> 136) ) 89 to +22 1–14 GCA CTG G– – gtgagt Intron 1 (1.67) A L (V) Exon 2 (166) 23–207 14–69 ccacag –TG TCC TCA TTT GTC AAG gtgtgc Intron 2 (1.80) (V) S S F V K Exon 3 (240) 208–458 70–149 ccctag ATC CTG CGC GAC AAA G– – gtgagt Intron 3 (5.38) I L R D K (A) Exon 3A (> 770) 208–978 70–268 ccctag ATC CTG CGC GTG GTT TGA xxxxxx Intron 3A (?) I LRVV* Exon 4 (141) 459–580 150–197 ctgcag –CC AAC AGA GAG AAT GC– gtgagt Intron 4 (0.38) (A) N R E N (A) Exon 5 (71) 581–662 198–218 ccacag – –C TCG GAA TAG CCC TG– gtaagg Intron 5 (0.09) (A) S E * Exon 6 (> 641) 663–1307 3¢ UT (b1) cttcag GCC CTG GGC Ó FEBS 2003 Cloning and characterization of Na + channel b 1B (Eur. J. Biochem. 270) 4765 shown in Fig. 1A. The open reading frame, designated b 1B , is related to the sodium channel b 1 subunit. Conserved motifs of the sodium channel b subunit family were also presented in the human sodium channel b 1B subunit, including a signal peptide sequence, the extracellular immunoglobulin fold domain and the C-terminal trans- membrane domain. The predicted peptide contained a hydrophobic N-terminal residue (1–16 residues) with sequences highly predictive of signal cleavage sites that would result in mature proteins initiating at amino acid 17 (alanine). The hydrophobic C-terminal region (residues 243–262) may serve as a transmembrane domain. The estimated protein molecular mass was  30.4 and 28.9 kDa before and after removing the signal peptide from the N terminus, respectively. The in vitro translated human b 1B subunit migrated with an apparent molecular mass of 30 kDa (with signal peptide) when analyzed by 8–20% SDS/PAGE (data not shown). Peptide sequence compar- ison revealed that the predicted peptide was 72% identical to both that of human (Fig. 1A) and rat sodium channel b 1 subunits and rat b 1A subunit (Fig. 1B). Like the rat b 1A subunit, the human b 1B subunit contained an N-terminal region (residues 1–149) of 100% identity to the b 1 subunit and a novel C-terminal region (residues 150–268) with an identity to the b 1 subunit of less than 17% (Fig. 1A). The C-terminal region of the human b 1B subunit was also significantly different from the rat b 1A and putative mouse b 1A subunits (The amino acid sequence of mouse b 1A subunit is deduced from mouse genomic sequence. The presence of such a splicing variant has not been confirmed by any experiment.) The C-terminal portion of human b 1B shares less than 33% and 36% peptide sequence identity with rat and mouse b 1A subunits, respectively, while the same region of rat and mouse b 1A shares at least 77% identity (Fig. 1B). A genomic organization study of the human sodium channel b 1 subunit gene, SCN1B [12], revealed that the gene spans  9 kb over six exons and five introns on chromo- some 19 (19q13.1-q13.2). BLAST searches of the human genomic database, using the cDNA sequence of human b 1B , revealed that the N-terminal region of the human b 1B subunit (residues 1–149) was encoded by exons 1–3, whereas the novel C-terminal region was encoded by the part of intron 3 adjacent to exon 3 (Fig. 1C and Table 1). As the site of divergence between the b 1 and b 1B subunit cDNAs was located precisely at the exon 3/intron 3 boundary of the SCN1B gene, the human sodium channel b 1B subunit should be considered as a splicing variant of the b 1 subunit via the extension of exon 3 to intron 3 (or partial intron 3 retention) with an in-frame stop codon. Tissue distribution of the human b 1B subunit Northern blot analysis, using a human b 1B specific probe, showed that the b 1B transcript is abundant in human brain and skeletal muscle (Fig. 2A), and present at a very low level Fig. 2. Northern blot analysis of the gene expression of human b 1B (A,B) and b 1 (C,D) subunits, using human b-actin mRNA level as the control (E,F). (A), (C) and (E) are human multiple tissue blots; (B), (D) and (F) are human brain II blots. The cDNA fragment encoding residues 217–268 of the human b 1B subunit, and the cDNA fragment encoding the human b 1 subunit from amino acids 150 to 218, were used as probes for detecting the messages of human b 1B and b 1 subunits, respectively. A 2 kb human b-actin cDNA fragment was used as the control probe. The blots were incubated at 42 °Covernightand washed with 0.2· NaCl/Cit/0.1% SDS at 65 °C for 2 h. Finally, the blots were exposed to X-ray film in a )80 °C freezer for 2–18 h.The same blots were used for all three probes in the order b 1B , b 1 and b-actin, after they were stripped at 68 °C for 15 min and reconstituted at room temperature for 15 min using the Strip-EZ TM removal kit provided by Ambion. 4766 N. Qin et al. (Eur. J. Biochem. 270) Ó FEBS 2003 in heart, placenta, lung, liver, kidney and pancreas. In human brain, the b 1B transcript was most abundant in the cerebellum, followed by the cerebral cortex and occipital lobe (Fig. 2B). The overall expression pattern of human b 1B was very similar to that of human b 1 (Fig. 2C,D), except that human b 1 was more abundant in cerebral cortex than in cerebellum. If the transcript of the human b 1B subunit is spliced only from exon 1 (111 bp), exon 2 (185 bp), exon 3 (250 bp) and either partial or entire intron 3 (5.3 kb), the calculated size of the transcript should be less than 6 kb. However, the major transcript of b 1B , as determined by Northern blot, is  7.5 kb. This suggests that an additional unidentified splicing event must be present to generate a longer 3¢ untranslated region, which needs to be identified by further experiments. In addition, a second transcript of the human b 1B ,of 1.5 kb, was observed in skeletal muscle. Expression of the novel b 1B subunit was further investi- gated by immunohistochemistry with affinity purified anti- b 1B (see Experimental procedures). The anti-b 1B was generated against a peptide derived from the retained intron 3 in the human cDNA clone. As shown in Fig. 3, immunohistochemical analyses revealed that the b 1B sub- unit was expressed in many different regions in the human brain, including cerebellar Purkinje cells (Fig. 3A), cortex pyramidal neurons, and many of the neuronal fibers throughout the brain (data not shown), consistent with the results of Northern blot analysis. Strong immunolabe- ling was also observed in human dorsal root ganglion (DRG) (Fig. 3C), in fibers (arrowheads) of the spinal nerve (Fig. 3D) and in cortical neurons (large arrowheads) and their processes (small arrowheads) (Fig. 3E). The specific b 1B labeling in the Purkinje cells (arrowhead) was abolished when the primary antibody was preabsorbed with the specific peptide. Functional expression of the human b 1B subunit with Na V 1.2 in Xenopus oocytes To explore the regulatory function of the human b 1B subunit, we injected cRNA of human b 1B ,aswellascRNA of the sodium channel pore forming subunit Na V 1.2, into Xenopus oocytes. As shown in Fig. 4A–D, the rates of activation and inactivation of the sodium current via Fig. 3. Immunohistochemical analysis of b 1B subunit expression in human tissues. (A) The presence of b 1B in Purkinje cells (large arrowheads) and in their processes (small arrowheads) of the cerebellum. (B) Specific b 1B labeling was abolished in the Purkinje cells (arrowhead) when the primary antibody was preabsorbed with the specific peptide. (C) Human b 1B was also detected in dorsal root ganglia (large arrowheads) as well as in the surrounding capsule cells (small arrowheads). (D) b 1B was present in fibers (arrowheads) of the spinal nerve. (E) b 1B was present in cortical neurons (large arrowheads) and their processes (small arrowheads). Bar ¼ 25 lm (A, B, D, E); 50 lm(C). Ó FEBS 2003 Cloning and characterization of Na + channel b 1B (Eur. J. Biochem. 270) 4767 Fig. 4. The effect of b 1B and b 1 subunits on the function and expression levels of the Na V 1.2 channel expressed in Xenopus oocytes. Representative sodium current traces from oocytes expressing the sodium channel a subunit, Na V 1.2, in the absence (A) and presence of b 1 (B) and b 1B (C) subunits. Sodium currents were evoked by 15 ms long depolarizing pulses (as indicated) from a holding potential of )80 mV. (D) The effect of b 1B and b 1 subunits on current time courses. Representative currents at )10mVinthepresenceorabsenceofb 1B or b 1 subunits are shown normalized to their individual peak value. (E) Inset: current-voltage relationship (I-V curve) for Na V 1.2 alone (s)andNa V 1.2 : hb 1B (1 : 5) (d). Sodium currents were evoked by 15 ms long depolarization steps, ranging from )60 to 80 mV, at 10 mV increments, from a holding potential of )100 mV. The peak magnitude of the currents, elicited by test depolarizations to the various potentials, were measured and used to construct I-V curves. Data represent average currents from a single batch of oocytes. Main panel: voltage dependence of the conductance (G-V curve). Data from the average G-V curves for Na V 1.2 alone (s), Na V 1.2 : hb 1B (d), and Na V 1.2 : hb 1 (d)werefittedtoG¼ G max /(1 + exp[–zÆ(V-V ½ )/25]) with parameters G max ,z,andV,asdescribedinthetext.CurveswerenormalizedbydividingbyG max . (F) Voltage dependence of inactivation (steady-state inactivation curves). Channels were inactivated by 100 ms conditioning pulses ranging from )140 to 20 mV, at 10 mV increments, then activated by a 15 ms test pulse to 0 mV (symbols as in part E: main panel). The relative fraction of channels available for activation was measured as the peak current during the test pulse to 0 mV. Data from individual oocytes were fitted by I ¼ I max /G ¼ G max /(1 + exp[–zÆ(V-V ½ )/25]) + I min and normalized by I m /I max obtained from the fit. All data points (E, F) correspond to the mean ± SEM of the averaged normalized currents for the number of oocytes indicated. (G) Effect of b 1B on the current amplitude of Na V 1.2. Each individual data point in the histogram represents the peak inward current for a single oocyte. Also shown are the mean (j) and standard deviation (bars) for each cRNA a:b ratio, and the sample size per cRNA combination is shown in parenthesis. Data are from five different batches of oocytes, each batch injected with cRNA for the a subunit alone or with the human b 1B subunitatratiosof1 :5and1:20.Unpairedt-test statistical analysis resulted in P-values of 0.056 (a vs. a:b 1B ; 1 : 5), 1.3e-5 (a vs. a:b 1B ;1:20)and0.035(a:b 1B 1:5vs.a:b 1B ;1:20). 4768 N. Qin et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Na V 1.2 did not change significantly in the presence or absence of the b 1B subunit, whereas, under the same conditions, the rate of inactivation was increased in the presence of the b 1 subunit. The effects of b 1B were further assessed by studying the current-voltage (I-V) relationships, the voltage-dependence of the conductance (G-V curve), and the voltage dependence of steady-state inactivation. Except for a minor negative shift (3–4 mV) in the voltage- dependence of activation (Fig. 4E), no significant effect of the human b 1B subunit on the regulation of Na V 1.2 sodium channel properties was observed (Fig. 4E,F, and Table 2). Under the same conditions, the human b 1 subunit also shifted the G-V relationship left, to a similar extent (Fig. 4E and Table 2), but caused a significant shift of the steady state inactivation curve by  10 mV, towards more negative potentials (Fig. 4F and Table 2). Although no significant modulatory effect of the b 1B subunit on channel kinetics and steady-state properties was observed, we found that the b 1B subunit increased the ionic current conducted by Na V 1.2 sodium channels (e.g. Fig. 4E, inset). At cRNA ratios of 1:5 and 1:20 (Na V 1.2 : b 1B ), the average (n ¼ 16–22) peak ionic current densities were increased by two- and threefold, respectively (Fig. 4G). Despite significant vari- ability in current densities within and between batches of oocytes, statistical analysis indicated that the difference between Na V 1.2 expressing oocytes and those expressing Na V 1.2 : b 1B at a ratio of 1 : 20 was significant (P < 0.0001). Discussion We report here the cloning and characterization of the human VGSC b 1B subunit. The human b 1B subunit is a novel splicing variant of the b 1 subunit via alternative intron 3 retention. The retained intron encodes a novel extracellular, a transmembrane, and an intracellular region, sharing little homology with the human and rat b 1 (17% identity) and the rat b 1A (33% identity) subunits. Although the novel b 1B subunit has a structure similar to other sodium channel b subunits, it exhibits regulatory properties in Xenopus oocytes that distinguish it from the b 1 and b 1A subunits. It is interesting that the only regulatory function of the b 1B subunit, observed in this study, was its ability to increase the sodium current density when coexpressed with the tetrodotoxin sensitive channel, Na V 1.2, in oocytes without affecting any of its voltage dependent properties. Several previous studies have shown that the b 1 subunit not only increases the levels of functional sodium channel on the cell surface, but that it also changes voltage dependent activa- tion and inactivation [3,4,13]. In the present study, we also observed that the simultaneous injection of b 1 with Na V 1.2 into oocytes resulted in an increase of the inactivation rate and a shift of the steady state inactivation curve to a more negative potential ( 10 mV), as well as an increase in ionic current amplitude (not shown), consistent with other studies in oocytes [3]. However, under the same conditions, the b 1B subunit had little effect on the properties of Na V 1.2. The increase in ionic currents induced by coexpression of the human b 1B could result from an increase in the number of channels present in the membrane, N o ,anincreaseinthe probability of opening of the channels, P o , and/or an increase in the single channel conductance, c o . Discrimin- ation among these options, however, requires evaluation of single channel parameters by other means (single channel recording or mean-variance analysis). The modulatory property of the b 1B subunit is also different from that of the b 1A subunit reported by Isom’s group. In their studies [7], the coexpression of b 1A with Na V 1.2 in Chinese hamster lung cells resulted in a 2.5-fold increase in the sodium current density, slightly shifted the steady state inactivation curve to a more positive potential (which also distinguishes it from the b 1 subunit) and had no effect on channel activation. However, we are unable to rule out the possibility that the different regulatory properties observed between rat b 1A and human b 1B results from the use of different expression systems in the two studies. Although b 1 , b 1A and b 1B have different effects on Na V 1.2 channel properties (b 1 affects both activation and inactiva- tion, b 1A affects inactivation only, and b 1B has no effect on either), the subunits all share a common regulatory prop- erty, i.e. they increase the sodium current density regardless of expression system. These results suggest that alteration of channel kinetics and steady state properties may be a function distinct from the increase in current density on the cell surface induced by b 1 subunits. The functional differ- ences of the b 1 , b 1A and b 1B subunits suggest that the C-terminal half of b 1 and its splicing variants play an important role in the modulation of sodium channel properties. Based on the sequence differences between b 1 and its splicing variants, there are three regions on the human b 1B subunit that may alter its regulatory properties (a) the additional extracellular region ( 90 residues in the human b 1B vs.  55 residues in rat b 1A ), (b) the transmem- brane domain, and (c) the intracellular region. The trans- membrane region of the human b 1B is located at the C terminus (241–262 residues) with five intracellular residues. This unique structure of the human b 1B subunit is very similar to that of the calcium channel a 2 d subunit, which also has six residues downstream from the transmembrane Table 2. Steady-state properties of the sodium channel Na V 1.2 in the presence or absence of the b 1B or b 1 subunit. Activation: G=G max /(1 + [exp()z * (V)V 1/2 )/25]) Inactivation: I=I max /(1 + [exp()z * (V)V 1/2 )/25]) + I min G max zV 1/2 (mV) I max zV 1/2 (mV) I min NaV 1/2 1.00 4.18 )9.94 1.00 )2.63 )36.11 )0.03 NaV 1/2 /b 1 1.00 4.19 )12.14 0.98 3.37 )48.46 )0.01 NaV 1/2 /b 1B 1.00 4.20 )10.92 1.00 )2.70 )37.75 )0.02 Ó FEBS 2003 Cloning and characterization of Na + channel b 1B (Eur. J. Biochem. 270) 4769 domain serving as an intracellular segment [14]. To date, no regulatory function related to the single transmembrane domain and the short, five-residue intracellular segment of the calcium channel a 2 d subunit has been reported, except that the transmembrane domain is essential for anchoring the protein into the membrane [14]. Therefore, the addi- tional extracellular region is probably responsible for the differences of regulatory functions between the b 1B and b 1 subunit or rat b 1A subunit. Recently, Meadows et al.[15] reported that the intracellular segment of the b 1 subunit is required for the interaction with the a subunit, probably a crucial step for the regulation of channel properties. The tissue distribution of the human b 1B subunit is similar to that of the human b 1 subunit. Its message was detected in skeletal muscle and in a variety of subregions in the brain (Figs 2 and 3). More interestingly, the human b 1B subunit is also expressed in DRG neuron and fibers (Fig. 3). It is well known that the number of functional sodium channels and magnitude of sodium currents are differentially changed following peripheral nerve injury [16–19]. Our observations of the existence of the b 1B subunit in human DRG, and its ability to increase sodium current density when coexpressed with Na V 1.2a in Xenopus oocytes, suggest that the human b 1B subunit may be another candidate useful for studying the mechanism of upregulation of functional sodium channels on the cell surface and increasing the rate of spontaneous firing in peripheral neurons after nerve injury. It will be interesting to determine whether the human b 1B subunit is up-regulated, and whether its up-regulation is correlated with the increase of sodium channel activity, in injured human DRG neuron. Acknowledgements We thank Drs Mike X. Zhu, Rich R. Ryan and Yi Liu for their critical discussion of the manuscript, Ms S. Yagel for her help in subcloning, and Ms Patti A. Reiser, Norah A. Gumula, Brenda M. Hertzog and Debbie Polkovitch for their histological and immunohistochemical expertise. References 1. Catterall, W.A. 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